Heparin and heparan sulphate oligosaccharides

ABSTRACT

Isolated heparin or heparan sulphate oligosaccharide fragments having a chain length of at least 10 saccharides and no more than 50 saccharides, which are capable of binding BMP2, are disclosed. Also disclosed is the use of the same heparin or heparan sulphate oligosaccharide fragments in kits and pharmaceutical compositions, and the use of the same heparan sulphate oligosaccharide fragments in the repair and/or regeneration of connective tissue and bones, and the treatment of wounds.

FIELD OF THE INVENTION

The present invention relates to heparin and heparan sulphateoligosaccharides, including heparin-type or heparan sulphate-typeoligosaccharides of defined chain length and particularly, although notexclusively, to such oligosaccharides that bind BMP2.

BACKGROUND

Bone morphogenetic proteins (BMPs) are numbers of the transforminggrowth factor-β (TGF-β) superfamily that play crucial roles in variousprocesses including mesoderm formation, neural patterning, skeletaldevelopment and limb formation (1, 2). More than 15 members of BMPs havebeen identified, and they play important roles in tissue repair andre-modeling processes after injuries (3-7). In animal models, severalrecombinant BMPs were reported to induce ectopic bone formation andenhance healing of critical-sized segmental bone defects. Clinicalstudies have shown that use of recombinant human BMPs is a safe andeffective alternative to autologous bone grafting. Recombinant BMP2 andBMP-7 are approved for human use in spinal fusion and recalcitrantlong-bone non-unions, respectively (4-7).

At the cellular level BMP signaling is initiated by binding two types ofspecific transmembrane serine/threonine kinase receptors namely type I(BMPR-I) and type II (BMPR-II) receptors (8). It has been shown that,BMP2 signaling results from binding to preaggregated receptor complexesrather than free receptors in the cell membrane (9). After ligandbinding, the type I receptors are activated by the ligand bound IIreceptors. The activated type I receptor then phosphorylates members ofthe Smad family of intracellular proteins—Smad 1, Smad 5, and Smad8—which in turn assemble into heteromeric complexes with Smad 4 thattranslocate into the nucleus to regulate transcription of target genes(10-12).

It is reported that, the in vivo and in vitro biological activities ofBMPs were positively and negatively regulated by a large number ofextracellular and cell surface sulphated polysaccharides such as heparinand heparan sulphate (HS) (8,13-20). BMP2 converts the differentiationpathway of C2C12 myoblasts into that of osteoblast lineage and heparinenhances BMP2 induced osteoblast differentiation in C2C12 myoblasts invitro (20-22). As with other proteins, it is believed that specificsize, and sulphated residues in the heparin/heparan sulphate chains bindto BMP2 and thereby modulate receptor-mediated signaling of thesemolecules.

Over the years, hundreds of HS-binding proteins have been identified,but how HS interacts with these proteins and affects their stability,concentration, conformation and activity is a fundamental question inbiology. Several studies have demonstrated that the binding of growthfactors to HS and thus giving rise to mitogenic activity happens onlywhen specific structural features are present within the HS chain (23).Such features include sulphation at specific positions within adisaccharide; 6-O sulphated N-sulphate glucosamine and 2-O sulphatediduronic acid residues are particularly important, and minimum bindingsequences are generally at least 5-6 disaccharides in length (24-26).The precise structures of HS that are involved in these interactionshave remained elusive. Information on minimal binding sequences on HS isvital to understanding the rules of HS-protein interaction and design ofHS mimics that can target proteins in human diseases. The minimal lengthand structural features of heparin/HS motifs have been identified inonly a few cases (27-30). The first well studied example for the minimallength of the HS motif required for ligand binding and activity was theheparin-derived pentasaccharide were sufficient to interact withantithrombin III to inhibit clotting factors thrombin (IIa) and factorXa (28-30). However, heparin-derived tetrasaccharide were sufficient tointeract with fibroblast growth factor-2 (FGF-2), but oligosaccharidesof degree of polymerization (dp) 10-12 are required for optimizing theproliferative activity of FGF-2. However, the structural sequencerequired for antithrombin III binding, where the most characteristicfeature is the unusual 3-O sulphate group on a 6-O sulphateN-sulphoglucosamine residue (31). Furthermore, the structuralcharacterization of a HS pattern that could bind FGF-2 has illustratedthe importance of continuous stretches of the disulphide disaccharideN-sulphoglucosamine-iduronate 2-O sulphate for highest binding ability(32). These examples clearly demonstrated that the minimaloligosaccharide length units and specific structural features of HSpattern required for either binding or biological activity are notstrictly related.

In this context, the characterization of the minimal HS unit andspecific sulphate group of HS that binds BMP2 is of importance in anattempt to clarify the molecular mechanism that underline therequirement of HS in the regulation of BMP2 activity. However, HS isextremely heterogeneous in sequence and size and the source is limited.Heparin is similar in structure to the sulphated regions of HS.Therefore, in this study we examine the relationship between minimumlength and specific sulphate group of heparin-derived oligosaccharides,which is required for binding to BMP2 and their capability to enhanceBMP2 induced osteoblast differentiation on C2C12 myoblasts.

Previously, we identified and isolated an heparan sulphate that bindsBMP2 with high affinity and showed that this HS has activity in therepair/regeneration of bone. Our work is reported in WO2010/030244 A1and U.S. Pat. No. 9,498,494.

SUMMARY OF THE INVENTION

In one aspect of the present invention an isolated heparin or heparansulphate oligosaccharide is provided, the isolated heparin or heparansulphate oligosaccharide having a chain length of at least 6 saccharidesand no more than 50 saccharides.

Other aspects and embodiments are described in the appended claims.

In some embodiments the oligosaccharide has a chain length of one of: atleast 8, at least 10, or at least 12 saccharides.

In some preferred embodiments the oligosaccharide comprises or consistsof a chain length of 8, 10 or 12 saccharides.

In some embodiments the oligosaccharide has a chain length of no morethan one of: 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35,34, 33, 32, 32, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11 or 10 saccharides.

In some embodiments the oligosaccharide has a chain length of one of 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49 or 50 saccharides.

In some embodiments the oligosaccharide is N-sulphated. This maycomprise N-sulphation of N-acetyl-D-glucosamine (GlcNAc) residues in theheparin or heparan sulphate oligosaccharide chain. Preferably at least80% of N-acetyl-D-glucosamine (GlcNAc) residues in the isolated heparinor heparan sulphate are N-sulphated. In some embodiments this may be oneof at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

In some embodiments the oligosaccharide is 6-O sulphated (O-sulphationat C6 of N-sulphoglucosamine (GlcNS) residues). Preferably at least 80%of N-sulphoglucosamine (GlcNS) residues in the heparin or heparansulphate oligosaccharide chain are 6-O-sulphated. In some embodimentsthis may be one of at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

Although the oligosaccharide may be 2-O sulphated (O-sulphation at C2 ofIdoA and GlcA) to a varying degree, in some embodiments the isolatedheparin or heparan sulphate is 2-O de-sulphated.

In some embodiments the oligosaccharide binds BMP2 protein, optionallywith a K_(D) of less than one of: 100 μM, 50 μM, 10 μM, 1 μM, 100 nM, 50nM, 10 nM or 1 nM.

Preparations or compositions comprising the isolated heparin or heparansulphate are provided.

In one aspect of the present invention a pharmaceutical composition ormedicament is provided comprising the isolated heparin or heparansulphate in accordance with the aspects described above. Thepharmaceutical composition or medicament may further comprise apharmaceutically acceptable carrier, adjuvant or diluent.

In another aspect of the present invention a composition comprising theisolated heparin or heparan sulphate according to any one of the aspectsabove and BMP2 protein is provided.

In one aspect of the present invention the isolated heparin or heparansulphate is provided for use in a method of treatment.

In a related aspect of the present invention the use of the isolatedheparin or heparan sulphate in the manufacture of a medicament for usein a method of medical treatment is provided.

In another aspect of the present invention a method of treatment isprovided, the method comprising the step of administering the isolatedheparin or heparan sulphate to a subject in need of treatment thereof.

In aspects of the invention concerning a method of medical treatment themethod of treatment may comprise a method of wound healing in vivo, therepair and/or regeneration of connective tissue, the repair and/orregeneration of bone and/or the repair and/or regeneration of bone in amammal or a human. In some preferred embodiments the method of treatmentcomprises the repair and/or regeneration of a broken bone. In someembodiments the method of treatment may comprise simultaneous orsequential administration of BMP2 protein.

In some embodiment a method of treating a bone fracture in a patient isprovided, the method comprising administration of a therapeuticallyeffective amount of the isolated heparin or heparan sulphate to thepatient. In some embodiments the method comprises administering theisolated heparin or heparan sulphate to the tissue surrounding thefracture. In some embodiments the method comprises injection of theisolated heparin or heparan sulphate to the tissue surrounding thefracture. In such methods the isolated heparin or heparan sulphate maybe formulated as a pharmaceutical composition or medicament comprisingthe isolated heparin or heparan sulphate and a pharmaceuticallyacceptable carrier, adjuvant or diluent.

In some embodiments the method may further comprise administering BMP2protein to the patient. In such methods the isolated heparin or heparansulphate and BMP2 protein may be formulated as a pharmaceuticalcomposition comprising the isolated heparin or heparan sulphate and BMP2protein and a pharmaceutically acceptable carrier, adjuvant or diluent.

In a further aspect of the present invention a biocompatible implant orprosthesis comprising a biomaterial and the isolated heparin or heparansulphate is provided. In some embodiments the implant or prosthesis iscoated with the isolated heparin or heparan sulphate. In someembodiments the implant or prosthesis is impregnated with the isolatedheparin or heparan sulphate.

In another aspect of the present invention a method of forming abiocompatible implant or prosthesis is provided, the method comprisingthe step of coating or impregnating a biomaterial with the isolatedheparin or heparan sulphate. In some embodiments the method furthercomprises coating or impregnating the biomaterial with BMP2 protein.

In some embodiments a method of treating a bone fracture in a patient isprovided, the method comprising surgically implanting a biocompatibleimplant or prosthesis, which implant or prosthesis comprises abiomaterial and the isolated heparin or heparan sulphate, into tissue ofthe patient at or surrounding the site of fracture.

In some embodiments the implant or prosthesis is coated with theisolated heparin or heparan sulphate. In some embodiments the implant orprosthesis is impregnated with the isolated heparin or heparan sulphate.In some embodiments the implant or prosthesis is further impregnatedwith BMP2 protein.

In yet a further aspect of present invention a kit of parts is provided,the kit comprising a predetermined amount of the isolated heparin orheparan sulphate and a predetermined amount of BMP2. The kit maycomprise a first container containing the predetermined amount of theisolated heparin or heparan sulphate and a second container containingthe predetermined amount of BMP2. The kit may be provided for use in amethod of medical treatment. The method of medical treatment maycomprise a method of wound healing in vivo, the repair and/orregeneration of connective tissue, the repair and/or regeneration ofbone and/or the repair and/or regeneration of bone in a mammal or ahuman. The kit may be provided together with instructions for theadministration of the isolated heparin or heparan sulphate and BMP2protein separately, sequentially or simultaneously in order to providethe medical treatment.

In a further aspect of the present invention products are provided, theproducts containing therapeutically effective amounts of:

-   -   (i) the isolated heparin or heparan sulphate; and    -   (ii) BMP2 protein;        for simultaneous, separate or sequential use in a method of        medical treatment. The method of medical treatment may comprise        a method of wound healing in vivo, the repair and/or        regeneration of connective tissue, the repair and/or        regeneration of bone and/or the repair and/or regeneration of        bone in a mammal or a human. The products may optionally be        formulated as a combined preparation for co-administration.

In a further aspect of the present invention there is provided a methodof designing a heparin or heparan sulphate, optionally a heparin orheparan sulphate for use in a method of treatment as described herein,the method comprising determining one or more of the chain length,sulphation pattern and saccharide (or disaccharide) content or sequenceof a heparin or heparan sulphate that has BMP2 binding activity.

In another aspect of the present invention there is provided a method ofmanufacturing, producing or preparing a heparin or heparan sulphate,optionally a heparin or heparan sulphate for use in a method oftreatment as described herein, the method comprising one or more of thefollowing steps:

-   -   (i) determining one or more of the chain length, sulphation        pattern and saccharide (or disaccharide) content or sequence of        a heparin or heparan sulphate that has BMP2 binding activity;    -   (ii) synthesising one or a plurality of heparin or heparan        sulphate oligosaccharides having a chain length, and/or        sulphation pattern and/or saccharide (or disaccharide) content        or sequence correlated with BMP2 binding activity;    -   (iii) formulating one or a plurality of heparin or heparan        sulphate oligosaccharides having a chain length, and/or        sulphation pattern and/or saccharide (or disaccharide) content        or sequence correlated with BMP2 binding activity as a        pharmaceutical composition or medicament.

DETAILED DESCRIPTION OF THE INVENTION

In some embodiments heparin or heparan sulphate oligosaccharides may beobtained by size fractionation of heparin or heparan sulphatepreparations. As such the heparin and heparan sulphate oligosaccharidesmay be fragments of larger heparin or heparan sulphate molecules.Suitable sources of heparin and heparan sulphate preparations for sizefractionation include commercially available heparin and heparansulphate preparations. For example, heparan sulphate preparations can beobtained during isolation of heparin from pig intestinal mucosa (e.g.available from Celsus Laboratories Inc., Sigma Aldrich, Iduron, UK).

Other suitable sources of heparin or heparan sulphate include heparin orheparan sulphate from any mammal (human or non-human), particularly fromthe kidney, lung or intestinal mucosa. In some embodiments the heparinor heparan sulphate is from pig (porcine) or cow (bovine) intestinalmucosa, kidney or lung.

In other embodiments the heparin and heparan sulphates oligosaccharidesmay be obtained by chemical synthesis of the desired oligosaccharidechain.

Heparin or heparan sulphate oligosaccharides or fragments according tothe present invention may be provided in isolated form or insubstantially purified form. This may comprise providing a compositionin which the heparin or heparan sulphate oligosaccharide component is atleast 80% heparin or heparan sulphate oligosaccharide, more preferablyone of at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

Glycosaminoglycans

As used herein, the terms ‘glycosaminoglycan’ and ‘GAG’ are usedinterchangeably and are understood to refer to the large collection ofmolecules comprising an oligosaccharide, wherein one or more of thoseconjoined saccharides possess an amino substituent, or a derivativethereof. Examples of GAGs are chondroitin sulphate, keratan sulphate,heparin, dermatan sulphate, hyaluronate and heparan sulphate.

Heparin

Heparin is a highly sulphated glycosaminoglycan. The most commondisaccharide unit in heparin is a 2-O-sulphated iduronic acid and6-O-sulphated, N-sulphated glucosamine, IdoA(2S)-GlcNS(6S). This makesup about 85% of heparins from beef lung and about 75% of those fromporcine intestinal mucosa. Although it is related to heparan sulphate,heparan sulphate differs from heparin in that heparan sulphate isnormally composed of a glucuronic acid (GlcA) linked toN-acetylglucosamine (GlcNAc) which makes up around 50% of the totaldisaccharide units.

Heparan Sulphate (HS)

Heparan sulphate proteoglycans (HSPGs) represent a highly diversesubgroup of proteoglycans and are composed of heparan sulphateglycosaminoglycan side chains covalently attached to a protein backbone.The core protein exists in three major forms: a secreted form known asperlecan, a form anchored in the plasma membrane known as glypican, anda transmembrane form known as syndecan. They are ubiquitous constituentsof mammalian cell surfaces and most extracellular matrices. There areother proteins such as agrin, or the amyloid precursor protein, in whichan HS chain may be attached to less commonly found cores.

“Heparan Sulphate” (“Heparan sulphate” or “HS”) is initially synthesisedin the Golgi apparatus as polysaccharides consisting of tandem repeatsof D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc). Thenascent polysaccharides may be subsequently modified in a series ofsteps: N-deacetylation/N-sulphation of GlcNAc, C5 epimerisation of GlcAto iduronic acid (IdoA), O-sulphation at C2 of IdoA and GlcA,O-sulphation at C6 of N-sulphoglucosamine (GlcNS) and occasionalO-sulphation at C3 of GlcNS. N-deacetylation/N-sulphation, 2-O-, 6-O-and 3-O-sulphation of HS are mediated by the specific action of HSN-deacetylase/N-sulphotransferase (HSNDST), HS 2-O-sulphotransferase(HS2ST), HS 6-O-sulphotransferase (HS6ST) and HS 3-O-sulphotransferase,respectively. At each of the modification steps, only a fraction of thepotential substrates are modified, resulting in considerable sequencediversity. This structural complexity of HS has made it difficult todetermine its sequence and to understand the relationship between HSstructure and function.

Heparan sulphate side chains consist of alternately arrangedD-glucuronic acid or L-iduronic acid and D-glucosamine, linked via (1→4)glycosidic bonds. The glucosamine is often N-acetylated or N-sulphatedand both the uronic acid and the glucosamine may be additionallyO-sulphated. The specificity of a particular HSPG for a particularbinding partner is created by the specific pattern of carboxyl, acetyland sulphate groups attached to the glucosamine and the uronic acid. Incontrast to heparin, heparan sulphate contains less N- and O-sulphategroups and more N-acetyl groups. The heparan sulphate side chains arelinked to a serine residue of the core protein through a tetrasaccharidelinkage(-glucuronosyl-β-(1→3)-galactosyl-β-(1→3)-galactosyl-β-(1→4)-xylosyl-β-1-O-(Serine))region.

Both heparan sulphate chains and core protein may undergo a series ofmodifications that may ultimately influence their biological activity.Complexity of HS has been considered to surpass that of nucleic acids(Lindahl et al, 1998, J. Biol. Chem. 273, 24979; Sugahara and Kitagawa,2000, Curr. Opin. Struct. Biol. 10, 518). Variation in HS species arisesfrom the synthesis of non-random, highly sulphated sequences of sugarresidues which are separated by unsulphated regions of disaccharidescontaining N-acetylated glucosamine. The initial conversion ofN-acetylglucosamine to N-sulphoglucosamine creates a focus for othermodifications, including epimerization of glucuronic acid to iduronicacid and a complex pattern of O-sulphations on glucosamine or iduronicacids. In addition, within the non-modified, low sulphated, N-acetylatedsequences, the hexuronate residues remain as glucuronate, whereas in thehighly sulphated N-sulphated regions, the C-5 epimer iduronatepredominates. This limits the number of potential disaccharide variantspossible in any given chain but not the abundance of each. Mostmodifications occur in the N-sulphated domains, or directly adjacent tothem, so that in the mature chain there are regions of high sulphationseparated by domains of low sulphation (Brickman et al. (1998), J. Biol.Chem. 273(8), 4350-4359, which is herein incorporated by reference inits entirety).

It is hypothesized that the highly variable heparan sulphate chains playkey roles in the modulation of the action of a large number ofextracellular ligands, including regulation and presentation of growthand adhesion factors to the cell, via a complicated combination ofautocrine, juxtacrine and paracrine feedback loops, so controllingintracellular signaling and thereby the differentiation of stem cells.For example, even though heparan sulphate glycosaminoglycans may begenetically described (Alberts et al. (1989) Garland Publishing, Inc,New York & London, pp. 804 and 805), heparan sulphate glycosaminoglycanspecies isolated from a single source may differ in biological activity.As shown in Brickman et al, 1998, Glycobiology 8, 463, two separatepools of heparan sulphate glycosaminoglycans obtained fromneuroepithelial cells could specifically activate either FGF-1 or FGF-2,depending on mitogenic status. Similarly, the capability of a heparansulphate (HS) to interact with either FGF-1 or FGF-2 is described in WO96/23003. According to this patent application, a respective HS capableof interacting with FGF-1 is obtainable from murine cells at embryonicday from about 11 to about 13, whereas a HS capable of interacting withFGF-2 is obtainable at embryonic day from about 8 to about 10.

As stated above HS structure is highly complex and variable between HS,indeed, the variation in HS structure is considered to play an importantpart in contributing toward the different activity of each HS inpromoting cell growth and directing cell differentiation. The structuralcomplexity is considered to surpass that of nucleic acids and althoughHS structure may be characterised as a sequence of repeatingdisaccharide units having specific and unique sulphation patterns at thepresent time no standard sequencing technique equivalent to thoseavailable for nucleic acid sequencing is available for determining HSsequence structure. In the absence of simple methods for determining adefinitive HS sequence structure HS molecules are positively identifiedand structurally characterised by skilled workers in the field by anumber of analytical techniques. These include one or a combination ofdisaccharide analysis, tetrasaccharide analysis, HPLC, capillaryelectrophoresis and molecular weight determination. These analyticaltechniques are well known to and used by those of skill in the art.

Two techniques for production of di- and tetra-saccharides from HSinclude nitrous acid digestion and lyase digestion. A description of oneway of performing these digestion techniques is provided below, purelyby way of example, such description not limiting the scope of thepresent invention.

Nitrous Acid Digestion

Nitrous acid based depolymerisation of heparan sulphate leads to theeventual degradation of the carbohydrate chain into its individualdisaccharide components when taken to completion.

For example, nitrous acid may be prepared by chilling 250 μl of 0.5 MH₂SO₄ and 0.5 M Ba(NO₂)₂ separately on ice for 15 min. After cooling,the Ba(NO₂)₂ is combined with the H₂SO₄ and vortexed before beingcentrifuged to remove the barium sulphate precipitate. 125 μl of HNO₂was added to GAG samples resuspended in 20 μl of H₂O, and vortexedbefore being incubated for 15 min at 25° C. with occasional mixing.After incubation, 1 M Na₂CO₃ was added to the sample to bring it to pH6. Next, 100 μl of 0.25 M NaBH₄ in 0.1 M NaOH is added to the sample andthe mixture heated to 50° C. for 20 min. The mixture is then cooled to25° C. and acidified glacial acetic acid added to bring the sample to pH3. The mixture is then neutralised with 10 M NaOH and the volumedecreased by freeze drying. Final samples are run on a Bio-Gel P-2column to separate di- and tetrasaccharides to verify the degree ofdegradation.

Lyase Digestion

Heparinise III cleaves sugar chains at glucuronidic linkages. The seriesof Heparinase enzymes (I, II and III) each display relatively specificactivity by depolymerising certain heparan sulphate sequences atparticular sulphation recognition sites. Heparinase I cleaves HS chainswith NS regions along the HS chain. This leads to disruption of thesulphated domains. Heparinase III depolymerises HS with the NA domains,resulting in the separation of the carbohydrate chain into individualsulphated domains. Heparinase II primarily cleaves in the NA/NS“shoulder” domains of HS chains, where varying sulphation patterns arefound. Note: The repeating disaccharide backbone of the heparan polymeris a uronic acid connected to the amino sugar glucosamine. “NS” meansthe amino sugar is carrying a sulphate on the amino group enablingsulphation of other groups at C2, C6 and C3. “NA” indicates that theamino group is not sulphated and remains acetylated.

For example, for depolymerisation in the NA regions using Heparinase IIIboth enzyme and lyophilised HS samples are prepared in a buffercontaining 20 mM Tris-HCL, 0.1 mg/ml BSA and 4 mM CaCl₂ at pH 7.5.Purely by way of example, Heparinase III may be added at 5 mU per 1 μgof HS and incubated at 37° C. for 16 h before stopping the reaction byheating to 70° C. for 5 min.

Di- and tetrasaccharides may be eluted by column chromatography.

Chemical Synthesis of Heparin or Heparan Sulphate Oligosaccharides

Synthetic heparin or heparan sulphate oligosaccarides of defined lengthmay be prepared using traditional solution phase chemistry withpublication of products either by crystallization of flash columnchromatography using Silicagel 60 (Fluka, Gillingham, UK).Oligosaccharides comprising 6 to 12 saccharide residues can be assembledfrom disaccharide precursors bearing protective groups. Final productscan be purified by size exclusion chromatography using Sephadex G-25(Sigma-Aldrich, Gillingham, UK) and lyophilised. Product structure andpurity can be confirmed by NMR spectroscopy and mass spectrometry

For example, the approach taken by Cole et al may be followed (Cole C L,Hansen S U, Baráth M, Rushton G, Gardiner J M, et al. (2010) SyntheticHeparan Sulphate Oligosaccharides Inhibit Endothelial Cell FunctionsEssential for Angiogenesis. PLoS ONE 5(7): e11644 doi:10 1371/journalpone 00011644).

Synthetic heparin or heparan sulphates may be prepared as analogues ofheparin or heparan sulphate oligosaccharides identified by sizefractionation of preparations containing long chain length heparin orheparan sulphate oligosaccharides, such as commercially availableheparin or heparan sulphate preparations from, e.g., porcine mucosa.

Synthetic heparin or heparan sulphate oligosaccharides may be preparedso as to have a specified chain length, sulphation pattern anddisaccharide content and the design of such oligosaccharides may bebased on information about chain length, sulphation pattern anddisaccharide content of heparin or heparan sulphate oligosaccaridesidentified by analysis or size fractionated preparations ofheterogeneous heparin or heparan sulphate preparations, as describedherein.

As such, in a further aspect of the present invention there is provideda method of designing a heparin or heparan sulphate, optionally aheparin or heparan sulphate useful in a method of treatment as describedherein, the method comprising determining one or more of the chainlength, sulphation pattern and saccharide (or disaccharide) content orsequence of a heparin or heparan sulphate that has BMP2 bindingactivity.

In another aspect of the present invention there is provided a method ofmanufacturing, producing or preparing a heparin or heparan sulphate,optionally a heparin or heparan sulphate useful in a method of treatmentas described herein, the method comprising one or more of the followingsteps:

-   -   (i) determining one or more of the chain length, sulphation        pattern and saccharide (or disaccharide), content or sequence of        a heparin or heparan sulphate that has BMP2 binding activity;    -   (ii) synthesising one or a plurality of heparin or heparan        sulphate oligosaccharides having a chain length and/or        sulphation pattern and/or saccharide (or disaccharide) content        or sequence correlated with BMP2 binding activity;    -   (iii) formulating one or a plurality of heparin or heparan        sulphate oligosaccharides having a chain length, and/or        sulphation pattern and/or saccharide for disaccharide content or        sequence co-related with BMP2 biding activity as a        pharmaceutical composition or medicament

HS3

HS3 is a BMP2 binding heparan sulphate, described in U.S. Pat. No.9,498,494 and in WO2010/030244 (where it is called HS/BMP2), bothincorporated herein in their entirety by reference.

HS3 is obtainable by methods of enriching mixtures of compoundscontaining one or more GAGs that bind to a polypeptide corresponding tothe heparin-binding domain of BMP2. The enrichment process may be usedto isolate HS3.

HS3 is believed to potentiate (e.g. agonize) the activity of BMP-2 andhence its ability to stimulate stem cell proliferation and boneformation.

In addition to being obtainable by the methodology described in U.S.Pat. No. 9,498,494 and in WO2010/030244, HS3 can also be definedfunctionally and structurally.

Functionally, HS3 is capable of binding a peptide having, or consistingof, the amino acid sequence of SEQ ID NO:1 (QAKHKQRKRLKSSCKRHP) or SEQID NO:2 (QAKHKQRKRLKSSCKRH), representing the heparin binding domain ofBMP2. Preferably, HS3 binds the peptide of SEQ ID NO:1 or 2 with a K_(D)of less than 100 μM, more preferably less than one of 50 μM, 40 μM, 30μM, 20 μM, or 10 μM.

Preferably, HS3 also binds BMP2 protein with a K₀ of less than 100 μM,more preferably less than one of 50 μM, 40 μM, 30 μM, 20 μM, or 10 μM.Binding between HS3 and BMP2 protein may be determined by the followingassay method.

BMP2 is dissolved in Blocking Solution (0.2% gelatin in SAB) at aconcentration of 3 μg/ml and a dilution series from 0-3 μg/ml inBlocking Solution is established. Dispensing of 200 μl of each dilutionof BMP2 into triplicate wells of Heparin/GAG Binding Plates pre-coatedwith heparin: incubated for 2 hrs at 37° C., washed carefully threetimes with SAB and 200 μl of 250 ng/ml biotinylated anti-BMP2 added inBlocking Solution. Incubation for one hour at 37° C., wash carefullythree times with SAB, 200 μl of 220 ng/ml ExtrAvidin-AP added inBlocking Solution, Incubation for 30 mins at 37° C., careful washingthree times with SAB and tap to remove residual liquid, 200 μl ofDevelopment Reagent (SigmaFAST p-Nitrophenyl phosphate) added. Incubateat room temperature for 40 minutes with absorbance reading at 405 nmwithin one hour.

In this assay, binding may be determined by measuring absorbance and maybe determined relative to controls such as BMP2 protein in the absenceof added heparan sulphate, or BMP2 protein to which an heparan sulphateis added that does not bind BMP2 protein.

The binding of HS3 is preferably specific, in contrast to non-specificbinding and in the context that the HS3 can be selected from otherheparan sulphates and/or GAGs by a method involving selection of heparansulphates exhibiting a high affinity binding interaction with thepeptide of SEQ ID NO:1 or 2 or with BMP2 protein.

HS3 according to the present invention preferably enhances BMP2 inducedAlkaline Phosphatase (ALP) activity in cells of the mouse myoblast cellline C2C12 to a greater extent than the enhancement obtained by additionof corresponding amounts of BMP2 protein or Heparin alone. Preferably italso enhances BMP2-induced ALP activity in C2C12 cells to a greaterextent than that induced by combined addition of corresponding amountsof BMP2 protein and heparin, or of BMP2 protein and a heparan sulphatethat does not bind BMP2 protein with high affinity.

Enhancement of ALP activity can be measured by performing the followingALP Assay. C2C12 cells are plated at 20,000 cells/cm² in a 24-well platein DMEM (e.g. Sigma-Aldrich Inc., St. Louis, MO) containing 10% FCS(e.g. Lonza Group Ltd., Switzerland) and antibiotics (1% Penicillin and1% Streptomycin) (e.g. Sigma-Aldrich Inc., St. Louis, MO) at 37° C./5%CO₂. After 24 hours, the culture media is switched to 5% FCS low serummedia containing different combinations of 100 ng/mL BMP2 (e.g. R&DSystems, Minneapolis, MN), 3 mg/mL Celsus HS and varying concentrationsof BMP2-specific (+ve HS) and non-specific (−ve HS) Celsus HSpreparations. Cell lysis is carried out after 3 days using RIPA buffercontaining 1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 2 mM EDTA,0.5% Igepal (NP40), 0.1% Sodium dodecyl sulphate (SDS) and 1% ProteaseInhibitor Cocktail Set III (Calbiochem. Germany). The protein content ofthe cell lysate is determined by using BCA protein assay kit (PierceChemical Co., Rockford, IL). ALP activity in the cell lysates was thendetermined by incubating the cell lysates with p-nitrophenylphosphatesubstrate (Invitrogen, Carlsbad, CA). The reading is normalized to totalprotein amount and presented as relative amount to the group containingBMP2 treatment alone.

Enhancement of ALP activity in C2C12 cells can also be followed byimmunohistochemical techniques, such as the following an ALP stainingprotocol. ALP Staining. C2C12 cells are cultured as described in theassay methodology immediately above. After 3 days of treatment, the celllayer is washed in PBS and stained using Leukocyte Alkaline PhosphataseKit (e.g. Sigma-Aldrich Inc., St. Louis. MO) according to manufacturer'sspecification. The cell layer is fixed in citrate buffered 60% acetoneand stained in alkaline-dye mixture containing Naphthol AS-MXPhosphatase Alkaline and diazonium salt. Nuclear staining is performedusing Mayer's Hematoxylin solution.

These techniques can be used to identify HS3 as a heparan sulphate thatenhances a greater degree of BMP2 protein induced ALP activity in C2C12cells compared with non-specific heparan sulphates, e.g. heparansulphates that do not bind BMP-2 protein.

HS3 prolongs the effects of BMP2 signalling to levels that equal orexceed those of heparin. This can be assessed by the following assay.C2C12 cells are exposed to (i) nothing, (ii) BMP2 alone, (iii)BMP2+Heparin or (iv) BMP2+HS3 for 72 hours and the phosphorylationlevels of the BMP2-specific intracellular signaling molecule Smad1/5/8are monitored by immunoblotting.

An important functional property of HS3 is its ability to enhance theprocess of bone repair, particularly in mammalian subjects. This may betested in a bone repair model, in which the speed and quality of bonerepair in control animals (e.g. animals not given HS or animals given anHS that does not bind BMP2 protein or the peptide of SEQ ID NO:1 or 2)and HS3 treated animals is compared. Speed and quality of bone repairmay be assessed by analysing generation of bone volume at the wound siteover time, e.g. by X-ray and microCT imaging analysis of the wound.

Recent research has shown that gamma-irradiation does not affect HS3binding affinity toward BMP2. Furthermore, irradiation did notsignificantly affect HS3's ability to synergistically enhance theosteogenic effects of BMP2. This confirmed that gamma-irradiation can beutilised for the sterilisation of HS3 products without affectingbiological activity. Therefore HS3 could be incorporated into orthoticimplants, scaffolds and other medical devices, that need to besterilised by such methods, for use in the treatment of a range ofdiseases and disorders [33].

Structurally, N-sulfation of N-acetyl-D-glucosamine (GlcNAc) residues inHS3 has been found to be important as regards maintaining bindingaffinity for BMP2 protein. N-desulfation was shown to lead to asignificant reduction in BMP2 protein binding affinity.

6-O-sulfation (O-sulphation at C6) of N-sulphoglucosamine (GlcNS)residues was also found to be of moderate significance as regardsmaintaining binding affinity for BMP2 protein. 6-O-desulfation led tosome reduction in BMP2 protein binding affinity.

2-O-sulfation (O-sulphation at C2) of IdoA and/or D-glucuronic acid(GlcA) residues was found not to affect BMP2 protein binding. As such,HS3 may optionally be either 2-O-sulfated or 2-O-desulfated.

The disaccharide composition of HS3 may be determined by digestion withheparin lyases I, II and III to completion and then subjecting theresulting disaccharide fragments to capillary electrophoresis analysis.

HS3 includes heparan sulphate that has a disaccharide composition within±10% (more preferably ±one of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or0.5%) of the values shown for each disaccharide in the table below, asdetermined respectively by lyase digestion and SAX-HPLC analysis ordigestion with heparin lyases I, II and III to completion and thensubjecting the resulting disaccharide fragments to capillaryelectrophoresis analysis.

Disaccharide Normalised weight percentage ΔHexUA,2SGlcNS,6S 14.8ΔHexUA,2S-GlcNS 4.9 ΔHexUA-GlcNS,6S 11.1 ΔHexUA,2SGlcNAc,6S 4.8ΔHexUA-GlcNS 22.2 ΔHexUA,2S-GlcNAc 1.1 ΔHexUA-GlcNAc,6S 10.1ΔHexUA-GlcNAc 31.1

The disaccharide composition of HS3 as determined by digestion withheparin lyases I, II and Ill to completion and then subjecting theresulting disaccharide fragments to capillary electrophoresis analysismay have a disaccharide composition according to any one of thefollowing:

Disaccharide Normalised weight percentage ΔHexUA,2SGlcNS,6S 14.8 ± 3.0ΔHexUA,2S-GlcNS  4.9 ± 2.0 ΔHexUA-GlcNS,6S 11.1 ± 3.0 ΔHexUA,2SGlcNAc,6S 4.8 ± 2.0 ΔHexUA-GlcNS 22.2 ± 3.0 ΔHexUA,2S-GlcNAc  1.1 ± 0.5ΔHexUA-GlcNAc,6S 10.1 ± 3.0 ΔHexUA-GlcNAc 31.1 ± 3.0 orΔHexUA,2SGlcNS,6S 14.8 ± 2.0 ΔHexUA,2S-GlcNS  4.9 ± 2.0 ΔHexUA-GlcNS,6S11.1 ± 2.0 ΔHexUA,2SGlcNAc,6S  4.8 ± 2.0 ΔHexUA-GlcNS 22.2 ± 2.0ΔHexUA,2S-GlcNAc  1.1 ± 0.5 ΔHexUA-GlcNAc,6S 10.1 ± 2.0 ΔHexUA-GlcNAc31.1 ± 2.0 or ΔHexUA,2SGlcNS,6S 14.8 ± 2.0 ΔHexUA,2S-GlcNS  4.9 ± 1.0ΔHexUA-GlcNS,6S 11.1 ± 2.0 ΔHexUA,2SGlcNAc,6S  4.8 ± 1.0 ΔHexUA-GlcNS22.2 ± 2.0 ΔHexUA,2S-GlcNAc  1.1 ± 0.5 ΔHexUA-GlcNAc,6S 10.1 ± 2.0ΔHexUA-GlcNAc 31.1 ± 3.0 or ΔHexUA,2SGlcNS,6S 14.8 ± 1.0 ΔHexUA,2S-GlcNS 4.9 ± 0.4 ΔHexUA-GlcNS,6S 11.1 ± 1.0 ΔHexUA,2SGlcNAc,6S  4.8 ± 0.6ΔHexUA-GlcNS 22.2 ± 3.0 ΔHexUA,2S-GlcNAc  1.1 ± 0.4 ΔHexUA-GlcNAc,6S10.1 ± 1.0 ΔHexUA-GlcNAc 31.1 ± 1.6 or ΔHexUA,2SGlcNS,6S  14.8 ± 0.75ΔHexUA,2S-GlcNS  4.9 ± 0.3 ΔHexUA-GlcNS,6S  11.1 ± 0.75ΔHexUA,2SGlcNAc,6S  4.8 ± 0.45 ΔHexUA-GlcNS  22.2 ± 2.25ΔHexUA,2S-GlcNAc  1.1 ± 0.3 ΔHexUA-GlcNAc,6S  10.1 ± 0.75 ΔHexUA-GlcNAc31.1 ± 1.2 or ΔHexUA,2SGlcNS,6S 14.8 ± 0.5 ΔHexUA,2S-GlcNS  4.9 ± 0 2ΔHexUA-GlcNS,6S 11.1 ± 0.5 ΔHexUA,2SGlcNAc,6S  4.8 ± 0.3 ΔHexUA-GlcNS22.2 ± 1.5 ΔHexUA,2S-GlcNAc  1.1 ± 0.2 ΔHexUA-GlcNAc,6S 10.1 ± 0.5ΔHexUA-GlcNAc 31.1 ± 0.8

Digestion of HS3 with heparin lyases I, II and Ill and/or capillaryelectrophoresis analysis of disaccharides may be preferably performed asdescribed in Example 10 of U.S. Pat. No. 9,498,494.

Digestion of HS preparations with heparin lyase enzymes may be conductedas follows: HS preparations (1 mg) are each dissolved in 500 μL ofsodium acetate buffer (100 mM containing 10 mM calcium acetate, pH 7.0)and 2.5 mU each of the three enzymes is added; the samples are incubatedat 37° C. overnight (24 h) with gentle inversion (9 rpm) of the sampletubes; a further 2.5 mU each of the three enzymes is added to thesamples which are incubated at 37° C. for a further 48 h with gentleinversion (9 rpm) of the sample tubes; digests are halted by heating(100° C., 5 min) and are then lyophilized; digests are resuspended in500 μL water and an aliquot (50 μL) is taken for analysis.

Capillary electrophoresis (CE) of disaccharides from digestion of HSpreparations may be conducted as follows: capillary electrophoresisoperating buffer is made by adding an aqueous solution of 20 mM H₃PO₄ toa solution of 20 mM Na₂HPO₄·12H₂O to give pH 3.5; column wash is 100 mMNaOH (diluted from 50% w/w NaOH); operating buffer and column wash areboth filtered using a filter unit fitted with 0.2 μm cellulose acetatemembrane filters; stock solutions of disaccharide Is (e.g. 12) areprepared by dissolving the disaccharides in water (1 mg/mL): calibrationcurves for the standards are determined by preparing a mix containingall standards containing 10 μg/100 μL of each disaccharide and adilution series containing 10, 5, 2.5, 1.25, 0.625, 0.3125 μg/100 μL isprepared; including 2.5 μg of internal standard (ΔUA,2S-GlcNCOEt.6S).The digests of HS are diluted (50 μL/mL) with water and the sameinternal standard is added (2.5 μg) to each sample. The solutions arefreeze-dried and re-suspended in water (1 mL). The samples are filteredusing PTFE hydrophilic disposable syringe filter units.

Analyses are performed using a capillary electrophoresis instrument onan uncoated fused silica capillary tube at 25° C. using 20 mM operatingbuffer with a capillary voltage of 30 kV. The samples are introduced tothe capillary tube using hydrodynamic injection at the cathodic (reversepolarity) end. Before each run, the capillary is flushed with 100 mMNaOH (2 min), with water (2 min) and pre-conditioned with operatingbuffer (5 min). A buffer replenishment system replaces the buffer in theinlet and outlet tubes to ensure consistent volumes, pH and ionicstrength are maintained. Water only blanks are run at both thebeginning, middle and end of the sample sequence. Absorbance ismonitored at 232 nm. All data is stored in a database and issubsequently retrieved and re-processed.

Duplicate or triplicate digests/analyses may be performed and thenormalized percentage of the disaccharides in the HS digest iscalculated as the mean average of the results for the analyses.

HS3 exhibits high affinity binding to BMP2 protein or the peptide of SEOID NO:1 or 2.

The structural differences of HS3 compared with heparan sulphates thatdo not bind BMP2 protein may also be illustrated by conducting surfaceplasmon resonance analysis. For example, the angle shift can be used todistinguish HS3 from other heparan sulphates.

Fragments of HS3

Some aspects and embodiments of the present invention concern fragmentsof HS3, or mixtures comprising fragments of HS3.

Preferably a fragment of HS3, is an oligosaccharide chain of HS3 thathas been truncated, cleaved or divided, e.g. by action of a lyase of anHS3 oligosaccharide chain to create more than one shorter chain.Preferred fragments are those that retain the ability to bind BMP2,enhance BMP2-mediated ALP activity, enhance BMP2 mediated Smad 1/5/9phosphorylation and/or enhance bone repair.

HS3 fragments and mixtures of HS3 fragments preferably exclude fulllength BMP2 binding heparan sulfate, such as HS3.

Full length HS3 typically has an average chain length of about 50saccharides or more, and an average molecular weight of about 15 kDa. Assuch, HS3 fragments and mixtures of HS3 fragments may have less than 10%oligosaccharide chains that have a chain length of greater than 50saccharides. Optionally, this may be a percentage selected from one of5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95%.

Mixtures of Heparin or Heparin Sulphate Oligosaccharides

Mixtures may comprise a plurality of oligosaccharide chains each of theplurality having defined length in terms of number or saccharides and,optionally, in terms of functional properties such as BMP2 binding.

The mixture may be heterogeneous in terms of the oligosaccharidecontent, for example it may contain oligosaccharides according to theinvention of varying length but all within a defined length range.

A mixture may contain other components, e.g. other glycosaminoglycans orheparin or heparan sulphate species. In some embodiments the mixturedoes not contain glycosaminoglycans, heparin or heparan sulphate speciesthat are different to the oligosaccharides according to the presentinvention.

Formulating Pharmaceutically Useful Compositions and Medicaments

In accordance with the present invention methods are also provided forthe production of pharmaceutically useful compositions, which may bebased on an identified heparin or heparan sulphate. Such methods ofproduction may further comprise one or more steps selected from:

-   -   (a) identifying and/or characterising the structure of a        selected heparin or heparan sulphate;    -   (b) obtaining the heparin or heparan sulphate;    -   (c) mixing the selected heparin or heparan sulphate with a        pharmaceutically acceptable carrier, adjuvant or diluent.

For example, a further aspect of the present invention relates to amethod of formulating or producing a pharmaceutical composition for usein a method of treatment, the method comprising:

-   -   (i) identifying and/or isolating the heparin or heparan        sulphate; and/or    -   (ii) formulating a pharmaceutical composition by mixing the        heparin or heparan sulphate, with a pharmaceutically acceptable        carrier, adjuvant or diluent.

Certain pharmaceutical compositions formulated by such methods maycomprise a prodrug of the selected substance wherein the prodrug isconvertible in the human or animal body to the desired active agent. Inother cases the active agent may be present in the pharmaceuticalcomposition so produced and may be present in the form of aphysiologically acceptable salt.

Biomaterials

Pharmaceutical compositions and medicaments of the invention may takethe form of a biomaterial that is coated and/or impregnated withisolated heparin or heparan sulphate. An implant or prosthesis may beformed from the biomaterial. Such implants or prostheses may besurgically implanted to assist in transplantation of cells.

The isolated heparin or heparan sulphate may be applied to implants orprostheses to accelerate new tissue formation at a desired location. Itwill be appreciated that heparins and heparan sulphates, unlikeproteins, are particularly robust and have a much better ability towithstand the solvents required for the manufacture of syntheticbioscaffolds and application to implants and prostheses.

The biomaterial may be coated or impregnated with the isolated heparinor heparan sulphate. Impregnation may comprise forming the biomaterialby mixing the isolated heparin or heparan sulphate with the constitutivecomponents of the biomaterial, e.g. during polymerisation, or absorbingthe isolated heparin or heparan sulphate into the biomaterial. Coatingmay comprise adsorbing the isolated heparin or heparan sulphate onto thesurface of the biomaterial.

The biomaterial should allow the coated or impregnated the isolatedheparin or heparan sulphate to be released from the biomaterial whenadministered to or implanted in the subject. Biomaterial releasekinetics may be altered by altering the structure, e.g. porosity, of thebiomaterial.

In addition to coating or impregnating a biomaterial with the isolatedheparin or heparan sulphate, one or more biologically active moleculesmay be impregnated or coated on the biomaterial. For example, at leastone chosen from the group consisting of: BMP-2, BMP-4, OP-1, FGF-1,FGF-2, TGF-β1, TGF-β2, TGF-β3; VEGF; collagen; laminin; fibronectin;vitronectin. In addition or alternatively to the above bioactivemolecules, one or more bisphosphonates may be impregnated or coated ontothe biomaterial along with the isolated heparin or heparan sulphate.Examples of useful bisphosphonates may include at least one chosen fromthe group consisting of: etidronate; clodronate; alendronate;pamidronate; risedronate; zoledronate.

The biomaterial provides a scaffold or matrix support. The biomaterialmay be suitable for implantation in tissue, or may be suitable foradministration (e.g. as microcapsules in solution).

The implant or prosthesis should be biocompatible, e.g. non-toxic and oflow immunogenicity (most preferably non-immunogenic). The biomaterialmay be biodegradable such that the biomaterial degrades. Alternatively anon-biodegradable biomaterial may be used with surgical removal of thebiomaterial being an optional requirement.

Biomaterials may be soft and/or flexible, e.g. hydrogels, fibrin web ormesh, or collagen sponges. A “hydrogel” is a substance formed when anorganic polymer, which can be natural or synthetic, is set or solidifiedto create a three-dimensional open-lattice structure that entrapsmolecules of water or other solutions to form a gel. Solidification canoccur by aggregation, coagulation, hydrophobic interactions orcross-linking.

Alternatively biomaterials may be relatively rigid structures, e.g.formed from solid materials such as plastics or biologically inertmetals such as titanium.

The biomaterial may have a porous matrix structure which may be providedby a cross-linked polymer. The matrix is preferably permeable tonutrients and growth factors required for bone growth.

Matrix structures may be formed by crosslinking fibres. e.g. fibrin orcollagen, or of liquid films of sodium alginate, chitosan, or otherpolysaccharides with suitable crosslinkers, e.g. calcium salts,polyacrylic acid, heparin. Alternatively scaffolds may be formed as agel, fabricated by collagen or alginates, crosslinked using wellestablished methods known to those skilled in the art.

Suitable polymer materials for matrix formation include, but are notlimited by, biodegradable/bioresorbable polymers which may be chosenfrom the group of: agarose, collagen, fibrin, chitosan,polycaprolactone, poly(DL-lactide-co-caprolactone),poly(L-lactide-co-caprolactone-co-glycolide), polyglycolide,polylactide, polyhydroxyalkanoates, co-polymers thereof, ornon-biodegradable polymers which may be chosen from the group of:cellulose acetate; cellulose butyrate, alginate, polysulphone,polyurethane, polyacrylonitrile, sulphonated polysulphone, polyamide,polyacrylonitrile, polymethylmethacrylate, co-polymers thereof.

Collagen is a promising material for matrix construction owing to itsbiocompatibility and favourable property of supporting cell attachmentand function (U.S. Pat. No. 5,019,087; Tanaka, S.; Takigawa, T.;Ichihara, S. & Nakamura, T. Mechanical properties of the bioabsorbablepolyglycolic acid-collagen nerve guide tube Polymer Engineering &Science 2006, 46, 1461-1467). Clinically acceptable collagen sponges areone example of a matrix and are well known in the art (e.g. from IntegraLife Sciences).

Fibrin scaffolds (e.g. fibrin glue) provide an alternative matrixmaterial. Fibrin glue enjoys widespread clinical application as a woundsealant, a reservoir to deliver growth factors and as an aid in theplacement and securing of biological implants (Rajesh Vasita, DhirendraS Katti. Growth factor delivery systems for tissue engineering: amaterials perspective. Expert Reviews in Medical Devices. 2006; 3(1):29-47; Wong C, Inman E, Spaethe R, Helgerson S. Thromb. Haemost. 200389(3): 573-582: Pandit A S, Wilson D J, Feldman D S. Fibrin scaffold asan effective vehicle for the delivery of acidic growth factor (FGF-1).J. Biomaterials Applications. 2000; 14(3): 229-242; DeBlois Cote M F,Doillon C J, Heparin-fibroblast growth factor fibrin complex: in vitroand in vivo applications to collagen based materials. Biomaterials.1994: 15(9): 665-672).

Luong-Van et al (In vitro biocompatibility and bioactivity ofmicroencapsulated heparan sulphate Biomaterials 28 (2007) 2127-2136),incorporated herein by reference, describes prolonged localised deliveryof HS from polycaprolactone microcapsules.

A further example of a biomaterial is a polymer that incorporateshydroxyapatite or hyaluronic acid.

Other suitable biomaterials include ceramic or metal (e.g. titanium),hydroxyapatite, tricalcium phosphate, demineralised bone matrix (DBM),autografts (i.e. grafts derived from the patient's tissue), orallografts (grafts derived from the tissue of an animal that is not thepatient). Biomaterials may be synthetic (e.g. metal, fibrin, ceramic) orbiological (e.g. carrier materials made from animal tissue, e.g.non-human mammals (e.g. cow, pig), or human).

The biomaterial can be supplemented with additional cells. For example,one can “seed” the biomaterial (or co-synthesise it) with stem cells.

In one embodiment the biomaterial may comprise be coated or impregnatedwith the isolated heparin or heparan sulphate, and further comprise BMP2(e.g. as a further coating or impregnated component) and cells, e.g.stem cells, adhered to the biomaterial.

Bone Fracture

In some aspects the present invention is concerned with the therapeuticuse (human and veterinary) of the isolated heparin or heparan sulphateto treat bone fracture. The isolated heparin or heparan sulphate isreported here to augment wound healing in bone. The isolated heparin orheparan sulphate stimulates bone regeneration following injury andcontributes to improved wound healing in bone. The isolated heparin orheparan sulphate provides improvements in the speed of bone fracturerepair enabling a reduction in the recovery time from injury.

Bone fracture is a medical condition. In this application “fracture”includes damage or injury to bone in which a bone is cracked, broken orchipped. A break refers to discontinuity in the bone. A fracture may becaused by physical impact, or mechanical stress or by medical conditionssuch as osteoporosis or osteoarthritis.

Orthopaedic classification of fractures includes closed or open andsimple or multi-fragmentary fractures. In closed fractures the skinremains intact, whilst in an open fracture the bone may be exposedthrough the wound site, which brings a higher risk of infection. Simplefractures occur along a single line, tending to divide the bone in two.Multi-fragmentary fractures spilt the bone into multiple pieces.

Other fracture types include, compression fracture, compacted fracture,spiral fracture, complete and incomplete fractures, transverse, linearand oblique fractures and comminuted fractures.

In most subjects bone healing (fracture union) occurs naturally and isinitiated following injury. Bleeding normally leads to clotting andattraction of white blood cells and fibroblasts, followed by productionof collagen fibres. This is followed by bone matrix (calciumhydroxyapatite) deposition (mineralisation) transforming the collagenmatrix into bone. Immature re-generated bone is typically weaker thanmature bone and over time the immature bone undergoes a process ofremodelling to produce mature “lamellar” bone. The complete bone healingprocess takes considerable time, typically many months.

Bones in which fractures occur and which may benefit from treatmentusing heparin or heparan sulphate oligosaccharide include all bonetypes, particularly all mammalian bones including, but not limited to,long bones (e.g. femur, humerus, phalanges), short bones (e.g. carpals,tarsals), flat bones (e.g. cranium, ribs, scapula, sternum, pelvicgirdle), irregular bones (e.g. vertebrae), sesamoid bones (e.g.patella).

Bones in which fractures occur and which may benefit from treatmentusing heparin or heparan sulphate oligosaccharide include skeletal bone(i.e. any bone of the skeleton), bones of the cranio-facial region,bones of the axial skeleton (e.g. vertebrae, ribs), appendicular bone(e.g. of the limbs), bone of the pelvic skeleton (e.g. pelvis).

Bones in which fractures occur and which may benefit from treatmentusing heparin or heparan sulphate oligosaccharide also include those ofthe head (skull) and neck, including those of the face such as the jaw,nose and cheek. In this respect, in some preferred embodiments heparinor heparan sulphate oligosaccharide may be used to assist in repair orregeneration of bone during dental or facial or cranial surgery, whichmay include reconstruction of bones (as distinct from teeth) of the faceand/or mouth, e.g. including the jawbone.

Bone fracture also includes pathological porosity, such as thatexhibited by subjects with osteoporosis.

Although not limiting to the present invention, the primary actions ofthe isolated heparin or heparan sulphate may be on cells within,adjacent to, or caused to migrate into the wound site and may be on thebone stem cells, the preosteoblasts or the osteoblasts, or on any of theancillary or vasculogenic cells found or caused to migrate into orwithin the wound bed.

The isolated heparin or heparan sulphate and pharmaceutical compositionsand medicaments comprising the isolated heparin or heparan sulphate areprovided for use in a method of treatment of bone fracture in amammalian subject.

Treatment may comprise wound healing in bone. The treatment may involverepair, regeneration and growth of bone. The isolated heparin or heparansulphate oligosaccharide facilitates fracture repair by facilitating newbone growth. The isolated heparin or heparan sulphate oligosaccharideacts to improve the speed of fracture repair enabling bone healing tooccur faster leading to improved recovery time from injury. Treatmentmay lead to improved bone strength.

Administration of heparin or heparan sulphate oligosaccharide ispreferably to the tissue surrounding the fracture. This may includeadministration directly to bone tissue in which the fracture hasoccurred. Administration may be to connective tissue surrounding thebone or fracture or to vasculature (e.g. blood vessels) near to andsupplying the bone. Administration may be directly to the site of injuryand may be to a callus formed by initial healing of the wound.

Medicaments and pharmaceutical compositions according to the presentinvention may be formulated for administration by a number of routes.Most preferably isolated heparin or heparan sulphate is formulated influid or liquid form for injection.

In some embodiments the isolated heparin or heparan sulphate isformulated as a controlled release formulation. e.g. in a drug capsulefor implantation at the wound site. The isolated heparin or heparansulphate may be attached to, impregnated on or soaked into a carriermaterial (e.g. a biomaterial) such as nanofibres or biodegradable paperor textile.

Pharmaceutical compositions, medicaments, implants and prosthesescomprising the isolated heparin or heparan sulphate oligosaccharide mayalso comprise BMP2. Owing to the ability of the isolated heparin orheparan sulphate oligosaccharide to bind BMP2, the isolated heparin orheparan sulphate oligosaccharide may act as a carrier of BMP2 assistingin delivery of BMP2 to the wound site and maintenance of BMP2 stability.

Administration is preferably in a “therapeutically effective amount”,this being sufficient to improve healing of the bone fracture comparedto a corresponding untreated fracture. The actual amount administered,and rate and time-course of administration, will depend on the natureand severity of the fracture. Prescription of treatment, e.g. decisionson dosage etc, is within the responsibility of general practitioners andother medical doctors, and will typically take account of the nature ofthe fracture, the condition of the individual patient, the site ofdelivery, the method of administration and other factors known topractitioners. Single or multiple administrations of isolated heparin orheparan sulphate doses may be administered in accordance with theguidance of the prescribing medical practitioner. Purely by way ofexample, isolated heparin or heparan sulphate may be delivered indosages of at least 1 ng/ml, more preferably at least 5 ng/ml andoptionally 10 ng/ml or more. Individual dosages may be of the order lessthan 1 mg and greater than 1 μg, e.g. one of about 5 μg, about 10 μg,about 25 μg, about 30 μg, about 50 μg, about 100 μg, about 0.5 mg, orabout 1 mg. Examples of the techniques and protocols mentioned above canbe found in Remington's Pharmaceutical Sciences. 20th Edition, 2000,pub. Lippincott. Williams & Wilkins.

Isolated heparin or heparan sulphate may be used to treat bone fracturealongside other treatments, such as administration of pain relieving oranti-inflammatory medicaments, immobilisation and setting of the bone,e.g. immobilising the injured limb in a plaster cast, surgicalintervention, e.g. to re-set a bone or move a bone to correctdisplacement, angulation or dislocation. If surgery is required isolatedheparin or heparan sulphate may be administered directly to (e.g.applied to) the fracture during the surgical procedure.

BMP2 Protein

In this specification BMP2 refers to Bone morphogenetic protein 2 (alsocalled bone morphogenic protein 2, BMP2 or BMP-2), which is a member ofthe TGF-β superfamily and is implicated in the development of bone andcartilage.

The amino acid sequence of bone morphogenetic protein 2 preprotein fromHomo sapiens can be found in GenBank under NCBI Accession No. NP_001191(NP_001191.1 GI:4557369) in which amino acids 1 to 23 represent thesignal peptide, and amino acids 24 to 396 represent the amino acidsequence of the proprotein.

In this specification “BMP2 protein” includes proteins having at least70%, more preferably one of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity with the amino acid sequence of the BMP2preprotein or BMP2 proprotein or with the amino acid sequence of themature BMP2 protein.

The BMP2 protein may be from, or derived from, any animal or human, e.g.non-human animals, e.g. rabbit, guinea pig, rat, mouse or other rodent(including from any animal in the order Rodentia), cat, dog, pig, sheep,goat, cattle (including cows, e.g. dairy cows, or any animal in theorder Bos), horse (including any animal in the order Equidae), donkey,and non-human primate or other non-human vertebrate organism; and/ornon-human mammalian animal; and/or human.

In this specification a subject to be treated may be any animal orhuman. The subject is preferably mammalian, more preferably human. Thesubject may be a non-human mammal (e.g. rabbit, guinea pig, rat, mouseor other rodent (including cells from any animal in the order Rodentia),cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, orany animal in the order Bos), horse (including any animal in the orderEquidae), donkey, and non-human primate). The non-human mammal may be adomestic pet, or animal kept for commercial purposes, e.g. a race horse,or farming livestock such as pigs, sheep or cattle. The subject may bemale or female. The subject may be a patient.

Methods according to the present invention may be performed in vitro orin vivo, as indicated. The term “in vitro” is intended to encompassprocedures with cells in culture whereas the term “in vivo” is intendedto encompass procedures with intact multi-cellular organisms.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The features disclosed in the foregoing description, or in the followingclaims, or in the accompanying drawings, expressed in their specificforms or in terms of a means for performing the disclosed function, or amethod or process for obtaining the disclosed results, as appropriate,may, separately, or in any combination of such features, be utilised forrealising the invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplaryembodiments described above, many equivalent modifications andvariations will be apparent to those skilled in the art when given thisdisclosure. Accordingly, the exemplary embodiments of the invention setforth above are considered to be illustrative and not limiting. Variouschanges to the described embodiments may be made without departing fromthe spirit and scope of the invention.

For the avoidance of any doubt, any theoretical explanations providedherein are provided for the purposes of improving the understanding of areader. The inventors do not wish to be bound by any of thesetheoretical explanations.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise” and “include”, andvariations such as “comprises”, “comprising”, and “including” will beunderstood to imply the inclusion of a stated integer or step or groupof integers or steps but not the exclusion of any other integer or stepor group of integers or steps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by theuse of the antecedent “about,” it will be understood that the particularvalue forms another embodiment.

All references mentioned above are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A to 1I. SPR competition binding assay sensorgrams. Representativesensorgrams generated from competitive binding experiments performed viaSPR. BMP-2 (25 nM) was pre-incubated with a variety of heparinoligosaccharides (5 μg or 10 μg) and applied to a heparin-derivatisedsurface. SPR sensorgrams for heparin (A), dp4 (B), dp6 (C), dp8 (D),dp10 (E), dp12 (F), de-2-O-sulfated heparin (G), de-6-O-sulfated heparin(H) and de-N-sulfated heparin (I) display variable reduction in responseover BMP-2 alone. All sensorgrams are representative of threeindependent experiments.

FIG. 2A to 2G. Chain length determines the ability of heparin to bindand stabilize BMP-2. Basic disaccharide structure of heparan sulfate andchondroitin sulfate (A). BMP-2 (20 ng) was incubated withheparin-Sepharose with or without glycosaminoglycans (B) or size definedheparin oligosaccharides (C) and bound BMP-2 detected via Western blot.(D) SPR sensorgram displaying the binding response generated by variousconcentrations (−6.25 nM, −12.5 nM, −25 nM, −50 nM, −100 nM) of BMP-2binding to a heparin coated surface. (E) SPR-based competitive bindingassays were performed by incubating heparin oligosaccharides (10 μg/mL)with BMP-2 (25 nM) and applying them to a heparin-coated SPR chip. (F,G) Differential scanning fluorimetry was performed by incubating BMP-2(5 μM) in the presence or absence of heparin oligosaccharides (50 μM;-PBS, -BMP-2 alone, -heparin, -dp4, -dp6, -dp8, -dp10, -dp12).Fluorescence generated from the binding of SYPRO orange dye to the coreof denatured BMP-2 was used to 17 determine relative complex stabilityvs. BMP-2+heparin. All data is representative of (B, C, D, F) orconstitutes mean±S.D. (E. G) of three independent experiments.****p<0.0001.

FIG. 3A to 3H. Size-exclusion chromatography of BMP-2 binding to heparinoligosaccharides. (A-D) 25 μM of each heparin oligosaccharides (dp6,dp8, dp10 or dp12) were eluted from a Superdex 200 column detected usingabsorption at 232 nm. (E-H) 25 μM BMP-2 and 25 μM of each heparinoligosaccharides (dp6, dp8, dp10 or dp12) were eluted from a Superdex200 column and detected using absorption at 232 nm. Chromatographs arerepresentative of three independent experiments.

FIG. 4A to 4E. BMP-2-induced Smad 1/5/9 phosphorylation, osteogenic genetranscription and ALP activity with heparin oligosaccharides. (A) C2C12cells were stimulated with or without BMP-2 (100 ng/mL) and heparinoligosaccharides (dp4, dp6, dp8, dp10, and dp12; 5 μg/mL) for up to 72h. Smad 1/5/9 phosphorylation was then detected via Western blot. C2C12cells were stimulated with BMP-2 in the presence or absence of heparinor heparin oligosaccharides for 3 days, after which osteogenic genetranscription (B-D) or ALP activity (E) was determined. Data arerepresented as the mean±S.D. of three independent experiments.****p<0.0001, ***p<0.001, **p<0.01. ns-p>0.05.

FIG. 5A to 5E. BMP-2 binding and thermal stability by desulfatedheparins. (A) The structure of the main repeating disaccharide unit inheparin/HS chains. (B) Competition of BMP-2 (20 ng) binding to heparinSepharose beads. (C) SPR-based competitive binding assays were performedby incubating selectively desulfated heparins (10 μg/mL) with BMP-2 (25nM) and applying them to a heparin-coated SPR chip. (D-E) Differentialscanning fluorimetry was performed by incubating BMP-2 (5 μM) with orwithout selectively desulfated heparins (50 μM) and SYPRO orange dye atincreasing temperature (-PBS, -BMP-2 alone, -heparin, -de-2-O-sulfated,-de-6-O-sulfated, -de-N-sulfated). Fluorescence generated from thebinding of SYPRO orange dye to the core of denatured BMP-2 was used todetermine relative complex stability vs. BMP-2+heparin. Data isrepresentative of (B. D) or constitutes mean±S.D. (C, E) of threeindependent experiments. ****p<0.0001; ns-p>0.05.

FIG. 6A to 6E. BMP-2-induced Smad 1/5/9 phosphorylation, transcriptionof osteogenic genes and ALP activity with selectively desulfatedheparins. (A) C2C12 cells were stimulated with or without BMP-2 (100ng/mL) and heparin or specific desulfated heparins (5 μg/mL) for up to72 h. Smad 1/5/9 phosphorylation was then detected via Western blot.(B-E) C2C12 cells were stimulated with or without BMP-2 (100 ng/mL) inthe presence or absence of heparin or desulfated heparin (5 μg/mL) andcultured for 3 days, after which osteogenic gene transcription (8-13) orALP activity (E) were determined. Data are represented as the means±S.D,of three independent experiments. ****p<0.0001, ***p<0.001, ns-p>0.05.

FIG. 7A to 7B. BMP-2-induced osteogenic differentiation andmineralization with heparin oligosaccharides and selectively desulfatedheparins. C2C12 cells were stimulated with or without BMP-2 (100 ng/mL)and heparin, heparin oligosaccharides or specific desulfated heparins (5μg/mL) for 12 days. (A) Cells were stained with Alizarin red to detectthe presence of calcium. (B) Alizarin red was extracted and quantifiedusing spectrophotometry (absorbance at 405 nm) and normalized to theBMP-2 alone treatment group. Data is representative of (A) orrepresented as (B) the mean±S.D. of three independent experiments.****p<0.0001, ns-p>0.05.

FIG. 8A to 8C. Establishment of controls for the rat ectopic boneformation assay. (A) Representative digital image, 2D x-rays and 3D μ-CTmicrographs of samples harvested from the hind limb muscle of rats. Thetreatments were collagen sponges alone (Ctrl; n=4); or with 5 μg BMP-2(BMP; n=4); or with 5 μg BMP-2+25 μg heparin (Hep; n=4). (B) The bonevolume measurements (mm3) for the respective samples as determined byμ-CT analyses. Results are expressed as mean±S.E.M. (C) Representativehistological sections showing the absence/presence of calcified bonematrix in the harvested samples. Staining consisted ofHematoxylin/Eosin, Modified Tetrachrome (blue: osteoid, red: bone) andvon Kossa/McNeals (black: calcified deposits). BM: Bone marrow, B: Bone,C: Calcified matrix, scale bars-100 μm. (For interpretation of thereferences to colour in this figure legend, the reader is referred tothe Web version of this article.)

FIG. 9A to 9C. The assessment of dps8, 10 and 12 in a rat ectopic boneformation assay. (A) Representative digital images, 2D x-rays and 3Dμ-CT micrographs of samples harvested from the hind limb muscle of rats.The treatments were collagen sponges with 5 μg BMP-2 and 25 μg of dps8(n=4), 10 (n=4) or 12 (n=5). (B) The bone volume measurements (mm3) forthe respective samples as determined by μ-CT analyses (dashed linerepresents bone volume for heparin treatment group). Results areexpressed as mean±S.E.M. (C) Representative histological sectionsshowing the presence of calcified bone matrix in the samples. Stainingconsisted of Hematoxylin/Eosin. Modified Tetrachrome (blue: osteoid,red: bone) and von Kossa/McNeals (black: calcified deposits). BM: Bonemarrow, B: Bone, C: Calcified matrix, scale bars-100 μm.

FIG. 10A to 10C. The assessment of de-2-O-, de-6-O- and de-N-sulfatedheparins in a rat ectopic bone formation assay. (A) Representativedigital image, 2D x-rays and 3D μ-CT micrographs of samples harvestedfrom the hind limb muscle of rats. The treatments were collagen spongeswith 5 μg BMP-2 and 25 μg of de-2-O- (n=4), de-6-O- (n=4) orde-N-sulfated (n=5) heparins. (B) The bone volume measurements (mm3) forthe respective samples as determined by p-CT analyses (dashed linerepresents bone volume for heparin treatment group). Results areexpressed as mean±S.E.M. (C) Representative histological sectionsshowing the presence of calcified bone matrix in the harvested samples.Staining consisted of Hematoxylin/Eosin, Modified Tetrachrome (blue:osteoid, red: bone) and von Kossa/McNeals (black: calcified deposits).BM: Bone marrow, B: Bone, C: Calcified matrix, scale bars-100 μm.

FIG. 11A to 11E. Binding, stabilization and activation of BMP-2 by othersulphated polysaccharides. (A) Representative SPR sensorgrams displayingthe binding response generated by various sulfated polysaccharides (10μg/mL) pre-incubated with BMP-2 (25 nM) and applied to aheparin-derivatised surface. (B) Normalized SPR data derived from (A)indicating the percentage of BMP-2 sequestered into solution by varioussulphated polysaccharides. (C) Differential scanning fluorimetry wasperformed by incubating BMP-2 (5 μM) in the presence or absence ofvarious sulfated polysaccharides (50 μM). (D) Fluorescence generatedfrom the binding of SYPRO orange dye to the core of denatured BMP-2 wasused to determine relative complex stability vs. BMP-2+heparin. (E)C2C12 cells were treated with BMP-2 (100 ng/mL) with or without varioussulphated polysaccharides (5 μg/mL) for 3 days, after which protein wasextracted and ALP activity was assessed. All data is representative of(A, C) or constitutes mean±S.D. (B, D-E) of three independentexperiments. **p<0.01, ***p<0.001, ****p<0.0001.

FIG. 12A to 12E. Binding, stabilization and activation of BMP-2 byde-N-sulfated/re-N-acetylated dp12. (A) Representative SPR sensorgramsdisplaying the binding response generated by heparin, heparin dp12 orde-N-sulfated/re-N-acetylated heparin dp12 (10 μg/mL) pre-incubated withBMP-2 (25 nM) and applied to a heparin-derivitised surface. (B)Normalized SPR data derived from (A) indicating the percentage of BMP-2sequestered into solution by heparin, heparin dp12 orde-N-sulfated/re-N-acetylated heparin dp12. (C) Differential scanningfluorimetry was performed by incubating BMP-2 (5 μM) in the presence orabsence of heparin, heparin dp12 and de-N-sulfated/re-N-acetylatedheparin dp12 (50 μM). (D) Fluorescence generated from the binding ofSYPRO orange dye to the core of denatured BMP-2 was used to determinerelative complex stability vs. BMP-2+heparin. (E) C2C12 cells weretreated with BMP-2 (100 ng/mL) with or without heparin, heparin dp12 orde-N-sulfated/re-N-acetylated heparin dp12 (5 μg/mL) for 3 days, afterwhich protein was extracted and ALP activity was assessed. All data isrepresentative of (A, C) or constitutes mean±S.D. (B, D-E) of threeindependent experiments. ***p<0.001, ****p<0.0001, ns-p>0.05.

FIG. 13 . Graph peaks relate to the crude size groups which wereseparated and pooled independently.

FIG. 14 . Relative ALP activity of heparin fragments and HS3 fragmentsof different lengths.

FIG. 15 . Sulfation analysis of HS3 fragments. Relative composition ofNAc disaccharides, N-sulphated disaccharides, 6-O-sulphateddisaccharides and 2-O-sulphated disaccharides in different size HS3fragments is demonstrated.

FIG. 16 . Disaccharide compositional differences between differentlength HS3 fragments shown in table form.

FIG. 17 . Disacharide composition of HS3 fragments with differentlengths is demonstrated.

FIG. 18 . Sulfation comparisons between heparin dp12 and HS3^(>dp36).Heparin dp12 and HS3^(>dp36) are the most biologically active fragmentsderived from heparin and HS3. Relative composition of NAc disaccharides,N-sulphated disaccharides, 6-O-sulphated disaccharides and 2-O-sulphateddisaccharides in different size HS3 fragments is demonstrated.

FIG. 19 . Disacharide composition comparison of Heparin dp12 andHS3^(>dp36), the most biologically active fragments derived from heparinand HS3.

FIG. 20 . Disacharide compositional comparisons between heparin dp12 andHS3>dp36. These are the most biologically active fragments of Heparinand HS3 and have different disaccharide compositions and shown in thetable.

EXAMPLES

In the following examples, the inventors describe the generation ofheparan sulfate and heparin oligosaccharides with variable sizes.Additionally, the inventors demonstrate that heparan sulfate and heparinoligosaccharides fragments with reduced chain lengths can function aseffectively as full length molecules.

Example 1: Methods to Determine Minimum Structural Requirements forBMP-2-Binding of Heparin Oligosaccharides

1.1. Materials

All cell culture reagents, chemicals and heparin were purchased fromSigma-Aldrich (U.S.A.). Recombinant BMP-2 was purchased from R&D SystemsInc. (U.S.A.). Smad 1/5/8 antibody was purchased from Santa CruzBiotechnology (Cat. #sc-6301-R; U.S.A.). Phospho-smad1/5/9 antibody(equivalent to phospho-smad1/5/8) was purchased from Cell SignalingTechnologies (Cat. #13820: U.S.A.). Actin antibody (Cat. #MAB1501R) andprotease inhibitor cocktail III were purchased from Merck-Millipore(U.S.A.). OIAzol lysis reagent was purchased from Qiagen (Germany).SYPRO orange. Superscript® Vilo™ cDNA synthesis kit, TaqMan® geneexpression assays and TaqMan® Fast universal PCR mastermix, Pierce BCAprotein assay kit, SuperSignal West Pico Chemiluminescent substrate,N-hydroxysuccinimide (NHS)-biotin and Zeba® spin 7 kDa MWCO desaltingcolumns were purchased from Thermo-Fischer (U.S.A.). Size-definedheparin oligosaccharides (dp4-dp20) and selectively desulfated heparinswere purchased from Iduron (U.K.). The C2C12 murine myoblast cell linewas purchased from the American Type Culture Collection (ATCC).Hyperfilm, Superdex 200 HR (10×300 mm) and Sensor Chip SA were purchasedfrom GE Healthcare (Sweden). Polycaprolactone (PCL) tubes (dimensions of4.5 mm inner diameter, 3 mm height and 1 mm wall thickness) werepurchased from Osteopore International Pte Ltd (Singapore). Collagensponges were obtained from Integra LifeSciences Corp (U.S.A.) andmeasured 7×21×5 mm. Each sponge was cut evenly into 6 pieces with asterile blade prior to implantation.

1.2. Surface Plasmon Resonance BMP-2 Competitive Binding Assay

Interactions between BMP-2 and heparin oligosaccharides were measuredvia competitive inhibition of BMP-2 binding to a heparin-coated sensorchip using surface plasmon resonance, as described by Lee et al [27].Briefly, 20 mg heparin was biotinylated at free amine groups using 8.6μM NHS-biotin in dimethyl sulfoxide, after which excess unreactedNHS-biotin was removed by passing the sample through two 7 kDa molecularweight cut-off Zeba spin desalting columns. Using the inbuiltimmobilization program on a Biacore T100 SPR system (GE healthcare), asensor chip SA (GE healthcare) was coated with heparin-biotin in HBS-EP0.05% buffer (150 mM NaCl, 10 mM HEPES, 3 mM EDTA, 0.05% (v/v) Tween-20,pH 7.4) at a flow rate of 30 μL/min until ˜40 response units (R.U.) wereachieved (where 1 R.U. is equal to −1 μg of heparin/mm2). Dosingexperiments were performed with BMP-2 with a range of concentrationsfrom 6.25 nM to 100 nM, after which a dose of 25 nM was selected forcompetitive inhibition experiments. Heparin oligosaccharides were usedat a concentration of 10 μg/mL in HBS-EP 0.1% buffer (HBS-EP with 0.1%(v/v) Tween-20). BMP-2 alone or BMP-2+heparin were applied to the chipfor 120 sat 30 μL/min, followed by a 600 s dissociation period of HBS-EP0.1% alone, after which the heparin surface was regenerated by twoinjections of 2 M NaCl in HBS-EP 0.1% for 60 s at 30 μL/min. Theresponse was measured as a function of time at 25° C. R.U.s for theheparin surface were determined by subtracting the R.U. of the control(uncoated) flow channel from those of the heparin-biotin-coated flowchannel. Representative sensorgrams from all competitive bindingexperiments may be found in supplementary information (FIG. 1 ). Resultswere normalized to the response of BMP-2 alone (100% response) andrepresented as the percentage reduction in response units vs. BMP-2alone. Data constitutes the mean±S.D. of three independent experiments.

1.3. Differential Scanning Fluorimetry/BMP-2 Thermal Stability Assay

The ability of heparin oligosaccharides to improve the thermal stabilityof BMP-2 was assessed via differential scanning fluorimetry (DSF).Briefly, BMP-2 (5 μM), heparin oligosaccharides (50 μM), urea (5 M), HCl(400 μM). SYPRO orange (×50) and PBS were gently mixed and aliquotedinto triplicate wells of a 96-well optical PCR plate (Axygen).Experiments were performed using a Quantstudio 6 real time PCR machine(Applied Biosciences) using the following heating cycle; 120 sincubation at 31° C. followed by 0.5° C. increases every 20 s to 81° C.Data was acquired using detection settings for TAMRA dye (γex 560 nm;γem 582 nm). BMP-2 melting temperature was determined by an observablepeak in fluorescence emission generated by the binding of SYPRO dye tothe core of the denatured protein. The highest temperatures determinedfrom the first derivative of the melt curve from each replicate wereselected and the relative stabilizing effect of each oligosaccharide vs.heparin was assessed using the following equation:

$\frac{{TmX} - {{TmBMP}2}}{{{Tm}{heparin}} - {{TmBMP}2}}$

Where Tm=melting temperature, X=oligosaccharide+BMP-2, BMP2=BMP-2 aloneand heparin=heparin+BMP-2). Data is represented as the mean±S.D. ofthree independent experiments.

1.4. Size-Exclusion Chromatography

A Superdex 200 HR column (300×10 mm, GE Healthcare) was equilibratedusing a Dionex ICS 3000 series High Performance Liquid Chromatography(HPLC) system at a flow rate of 0.5 ml/min with running buffer (10 mMHEPES, pH 7.2, and 150 mM NaCl). Heparin fragments (25 μM) wereincubated in the presence or absence of BMP-2 (25 μM) for 15 min at roomtemperature in running buffer, after which samples were loaded onto thecolumn and eluted under isocratic flow at 0.5 ml/min. Elution of sampleswas monitored via absorbance at 232 nm (heparin) and 280 nm (BMP-2). Allchromatographs are representative of three independent experiments.

1.5. Detection of Phosphorylated Forms of Smad 1/5/9 by Western Blotting

C2C12 murine myoblast cells were cultured in DMEM, 10% FCS and 100 U/mLpenicillin/streptomycin at 37° C. in 5% CO2. For experiments, cells wereseeded at 2×104 cells/cm2 in tissue culture treated 12-well plates.After 24 h, cells were washed and media was replaced with treatmentmedia (maintenance media with 5% FCS) with or without BMP-2 (100 ng/mL)and heparin, heparin oligosaccharides or desulfated heparins (5 μg/mL).At the indicated time points (24, 48 and 72 h), cells were washed withPBS and lysed with RIPA buffer in the presence of protease inhibitors(Merck-Millipore). Total protein was quantified using the BCA assay andsubsequently separated by SDS-PAGE (5 μg total protein per condition).Proteins were then transferred to nitrocellulose membranes and blockedfor 1 h in blocking buffer (Tris-buffered saline containing 5% (w/v)non-fat dry milk and 0.05% (v/v) Tween 20). Incubation with primaryantibodies diluted at 1:1000 (pSmad 1/5/9), 1:500 (Smad 1/5/8) or 1:5000(actin) was carried out overnight at 4° C. in blocking solution.Membranes were washed three times with Tris-buffered saline containing0.05% (v/v) Tween 20 and incubated with IgG-specificperoxidase-conjugated secondary antibodies, diluted 1:10000 in blockingbuffer. Blots were then washed and developed using SuperSignal West PicoChemiluminescent substrate (Thermo Scientific) on Hyperfilm (GEHealthcare) and scanned. Blots are representative of three independentexperiments.

1.6. Analysis of Osteogenic Transcript Expression Via Quantitative RealTime PCR

C2C12 cells were maintained and seeded as described above. Cells weretreated with or without BMP-2 (100 ng/mL) with heparin, heparinoligosaccharides, or selectively desulfated heparins (5 μg/mL). Thecells were cultured in treatment media for 3 days, after which total RNAwas harvested using OIAzol lysis reagent (Qiagen) according tomanufacturers instructions. Total RNA was quantified via Nanodrop 2000and 1 μg total RNA was reverse transcribed to generate complementary DNA(cDNA) using SuperScript Vilo (Thermo-Fisher) according tomanufacturer's instructions. Quantitative real-time PCR (qPCR) wasperformed using a Quantstudio 6 real-time PCR machine, using 40 ng ofcDNA per reaction in conjunction with TaqMan® gene expression assays(Thermo-Fisher). Transcription of osteogenesis-associated genes ofinterest were analyzed using the following TaqMan® gene expressionassays: alpl (Mm00475834_m1), sp7 (Mm04209856_m1) and runx2(Mm00501584_m1). Ribosomal RNA 18s (Mm04277571_s1) was used as aninternal control. Data was analyzed using the ΔΔCt method and normalizedto treatment with BMP-2 alone. Data is expressed as mean foldchange±S.D. of three independent experiments.

1.7. Alkaline Phosphatase Activity Assay

C2C12 cells were maintained as described above and seeded at 2×104cells/cm2 in tissue culture treated 24-well plates. The cells werecultured in treatment media for 3 days, after which total protein wasextracted and ALP activity was determined. Briefly, cells were washedwith PBS and total protein extracted with RIPA buffer containing aprotease inhibitor cocktail. Total protein concentration was determinedusing the BCA assay, then 7.5 μg of total protein per condition wascombined with p-nitrophenylphosphate (Thermo-Fisher). Absorbance wasmeasured at 405 nm via spectrophotometry and expressed as relative ALPactivity normalized to the BMP-2 alone treatment group. Data representsmean±S.D. of three independent experiments.

1.8. Osteogenic Differentiation and Mineralization

C2C12 cells were maintained as described above and seeded at 5×103cells/cm2 in tissue culture treated 12-well plates. Cells were culturedin osteogenic media (DMEM low glucose, 5% (v/v) FCS, 100 U/mLpenicillin/streptomycin, 10 mM β-glycerophosphate and 50 μg/mL ascorbicacid 2-phosphate) with or without BMP-2 (100 ng/mL) and heparin variants(5 μg/mL) for 12 days, with media being replenished every three days.Cells were washed with PBS and fixed with ice cold methanol for 20 minat −20° C. After fixation, cells were washed with water and stained withAlizarin red solution (0.1% (w/v), pH 4.3) for 20 min at roomtemperature with gentle agitation. Cells were then washed three timeswith water and imaged using a scanner. Alizarin red dye was extractedwith 10% (Of) acetic acid, neutralized with 10% (w/v) ammonium hydroxideand quantified using spectrophotometry (absorbance at 405 nm). Imagesare representative of at least three independent experiments andquantified Alizarin red stain represents mean±S.D. of three independentexperiments.

1.9. Ectopic Bone Formation Assay

Thirteen female, Sprague Dawley rats weighing 120-150 g had four hindlimb muscle pockets surgically created, two per limb [48]. Each pocketwas randomly filled with one of the following treatments; (1)Polycaprolactone tube (PT), (2) PT+collagen sponge (CS) (Ctrl), (3)PT+CS+BMP-2 (5 kg), (4) PT+CS+heparin (25 μg)+BMP-2 (5 μg). (5)PT+CS+dp8 (25 μ9)+BMP-2 (5 μg), (6) PT+CS+dp10 (25 μg)+BMP-2 (5 kg), (7)PT+CS+dp12 (25 kg)+BMP-2 (5 μg), (8) PT+CS+de-2-O sulfated heparin (25μg)+BMP-2 (5 μg) (9) PT+CS+de-6-O sulfated heparin (25 μg)+BMP-2 (5 kg,(10) PT+CS+de-N-sulfated heparin (25 μ9)+BMP-2 (5 μg). There were 4replicates for groups (1-3) and 5 replicates for groups (4-10). Thedoses and ratio of BMP-2 and heparin were based on those used inprevious studies [22, 32]. Rats were sacrificed and specimens harvested8 weeks post-implantation. All samples were assessed with 2D x-rays,μ-CT and histology to assess the extent of bone mineral deposition.

1.10. Surgical Methods

All surgical procedures were performed under aseptic conditions and instrict accordance to the guidelines of A*STAR's Institutional AnimalCare and Use Committee. General anesthesia was established by theadministration of isoflurane, Two transverse incisions, 1 cm each, weremade over each hind limb muscle. Pockets were then created by bluntdissection of the muscle, parallel to the longitudinal-axis of musclefibers. The specimens were then implanted into these pockets. Theincisions were then closed in both the muscle and skin layers.Prophylactic antibiotics (Baytril, 10 mg/kg) and analgesics(Buprenorphine, 0.01-0.05 mg/kg) were administered subcutaneously forthree days post-operation.

1.11. 2D Radiographic Analysis

2D images of muscle pockets were captured immediately post-operation andat week 8 using an Imaging Radiographic System (MUX-100, Shimadzu).Digital micrographs were subsequently generated from the x-rays.

1.12. 3D μ-CT Analysis

A μ-CT scanner (Skyscan 1076: Skyscan, Belgium) was utilized to scan theharvested specimens (resolution=35 μm; scanning width=68 mm) as per ourprevious methods [34, 35]. Scanner voltage and current were set to 104kV and 98 μA respectively. Cone-Beam CT-reconstruction® A Sasov software(Skyscan) was used to process the isotropic slice data and convert into2D images. Mimics 13.1 software (Materialise. Belgium) was then used toanalyze and remodel data in 3D, using the same number of slices andcylindrical region of interest (ROI) —for each specimen. The total bonevolume within the ROI was quantified by assigning a pre-determinedthreshold for total bone content. Specimens containing BMP-2, whichexhibited zero bone growth via x-ray and μ-CT (≤0.05 mm³) were excludedfrom further analysis. Data represents mean±S.E.M. of total bone volume(mm3).

1.13. Histological Evaluation

Specimens were fixed in 10% formalin (neutral buffered) under vacuum forone week. For paraffin histology, specimens were decalcified for twoweeks in 30% formic acid at room temperature. The specimens were thenprocessed with a vacuum infiltration processor (Sakura Finetek, Japan)using a 14 h program, followed by dehydration, clearing, and finallyembedding in Paraplast® paraffin wax (Thermo Scientific). Paraffinblocks were sectioned longitudinally at 5 μm using a rotary microtome(Leica Microsystems, Germany), and left to dry on positively-chargedmicroscope slides. These slides were subsequently stained withhematoxylin & eosin and modified tetrachrome. Both stains allow for theidentification of bone marrow and bone based on its morphology andcellular details. A blue stain and red patches represent osteoid andbone deposition respectively for the modified tetrachrome method. Forresin histology, specimens were dehydrated through an ethanol series,followed by processing and embedding in methylmethacrylate. Transversesections of 5 μm's were cut and stained with von Kossa/MacNeals, for thepositive identification of calcified deposits (which stained black). Allsections were examined with an Olympus Stereo (SZX12) and upright (BX51)fluorescence microscopes.

1.14. Statistical Methods

All data is represented as the mean±S.D. of at least three independentexperiments unless otherwise stated. For statistical analysis, one andtwo-way analysis of variance (ANOVA) with Tukey's post hoc analysis wasperformed using Prism 7 software (GraphPad, San Diego, USA). P<0.05 wasconsidered significant. The following symbols were used to indicatep-value range; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; ns—notsignificant (p>0.05).

Example 2: Minimum Structural Requirements for BMP-2-Binding of HeparinOligosaccharides—Results

2.1. Effect of Heparin Oligosaccharide Length on Competitive Binding ofBMP-2 to Heparin

To assess the minimum saccharide length requirement for binding ofheparin to BMP-2, we subjected heparin oligosaccharides (dp4, dp6, dp8,dp10, dp12, dp14, dp16, dp18 or dp20: the structure of a dp2 isillustrated in FIG. 2A) to SPR competitive binding assays. When a rangeof doses of BMP-2 were applied to a heparin-coated SPR chip, abundantresponse was observed (FIG. 2B). Pre-incubation with heparin resulted in97% of the BMP-2 being sequestered in solution, preventing binding tothe heparin-coated surface (FIG. 2 C). Dp4 were unable to sequesterBMP-2 into solution, suggesting they are incapable of preventing BMP-2from binding the heparin-coated surface. When dp6 and dp8 werepre-incubated with BMP-2 prior to application to the heparin-coatedchip, a reduction in R.U. was observed (FIG. 2C). Dp6 and dp8sequestered 4% and 24% of BMP-2 respectively (FIG. 2C). Dp10, dp12, dp14and dp16 further sequestered BMP-2 in solution, binding 36%, 61%, 76%and 80% respectively (FIG. 2C). Finally, dp18 and dp20 sequestered 85%and 82% of BMP-2 in solution respectively, quantities comparable tothose sequestered by heparin (p>0.05).

2.2. Heparin Oligosaccharides of Increasing Length Enhance the ThermalStability of BMP-2

We further investigated the interaction between heparin oligosaccharidesand BMP-2 by performing DSF, an assay which enabled us to investigatethe effect of heparin oligosaccharides on the thermal stability ofBMP-2. FIG. 20 displays the shift in peak fluorescence toward a highertemperature upon the addition of oligosaccharides of increasing size(FIG. 2D). FIG. 2E displays the relative increase in the thermalstability of BMP-2 with the addition of native heparin and heparinoligosaccharides ranging between dp4 and dp20. Dp4 and dp8 offeredminimal enhancement in BMP-2 thermal stability, whereas dp8 and dp10enhance BMP-2 thermal stability to half that achieved by native heparin(FIG. 2E). Dp12 offered a further increase in thermal stability over thesmaller fragments, and further increases in stability were observed fordp14, dp16 and dp18. Dp20 offered further increases in thermalstability, comparable with native heparin. Statistical analysis usingone-way ANOVA revealed that oligosaccharide size had a significanteffect on BMP-2 thermal stability (p<0.0001). Further post hoccomparisons between individual groups indicated that the stabilizingeffect of dp4-dp18 on BMP-2 was significantly less than that of nativeheparin (p<0.0001), with only a dp20 offering comparable stability toheparin (p>0.05).

2.3. Size-Exclusion Chromatography of BMP-2 and Heparin Oligosaccharides

We next investigated the ability of oligosaccharides to bind BMP-2 usinga size-exclusion chromatographic technique. FIG. 3 (A-D) displays thedp6-12 peak elution positions. After incubation of heparinoligosaccharides, it is evident that the elution position of dp6 or dp8saccharides remained unaffected by BMP-2 (FIG. 3E-F); indicating thatminimal binding occurred between BMP-2 and dp6 or dp8 fragments. Incontrast, the elution position of the dp10 saccharide was split equallyinto BMP-2-bound and -unbound peaks (FIG. 3G), again indicating this isthe minimal heparin length required for BMP-2 binding. When BMP-2 wasincubated with the dp12 preparation, the elution position of the singlemajor peak was significantly shifted (FIG. 3H). These data confirmedthat dp10 is the minimal heparin oligosaccharide length required forsignificant levels of BMP-2 binding, and dp12 for maximal levels. Forsubsequent in vitro studies, dp4 to dp12 were used.

2.4. Heparin Oligosaccharide Length Influences BMP-2-Induced Signalingand Differentiation of C2C12 Cells

We next examined the time course changes induced by BMP-2 andoligosaccharides (dp4-dp12) in the phosphorylation of the specificdownstream BMP-2 intracellular effector Smad 1/5/9. In the absence ofadditional heparin oligosaccharides, the levels of BMP-2-induced Smad1/5/9 phosphorylation were detectable at 24 and 48 h; after 72 h, Smad1/5/9 phosphorylation was no longer detectable in cultures exposed toBMP-2 alone (FIG. 4Ai-iii). Smad 1/5/9 phosphorylation levels weresimilar across all treatment groups at 24 and 48 h (FIG. 4Ai-ii). At 72h, Smad 1/5/9 phosphorylation had decreased progressively in culturestreated with shorter oligosaccharides (dp4, dp6 or dp8; FIG. 4Aiii).Cultures treated with heparin or dp12 fragments displayed the highestlevels of Smad 1/5/9 phosphorylation (FIG. 4Aiii). These data confirmthat oligosaccharides ≥dp10 in length prolong and maintain BMP-2-inducedSmad 1/5/9 phosphorylation. We next compared the bioactivity of heparinoligosaccharides by assaying BMP-2-induced up-regulation of osteogenicgene transcripts and ALP activity in C2C12 myoblast cells. We chose toassess changes in transcript level of alpl, runx2 and sp7, as all aremarkers of osteogenic differentiation. Additionally we chose to assessALP activity as ALP is a key marker of osteogenic differentiation.Alone, heparin and heparin oligosaccharides did not induce expression ofosteogenic genes alpl, sp7 and runx2, or ALP activity (FIG. 4B-E).However, when cells were exposed to 100 ng/ml of BMP-2, in the presenceof heparin, a rapid induction of osteogenic gene transcription and ALPactivity was observed (FIG. 4B-E). Dp4 and dp6 failed to substantiallyupregulate BMP-2-induced gene transcription or enhance ALP activity overBMP-2 alone (FIG. 4B-E). Dp8 upregulated gene transcription and enhancedALP activity, but to a lesser extent than native heparin (FIG. 4B-E).However, dp10 enhanced BMP-2-induced ALP activity to the levels ofnative heparin (FIG. 4E, p>0.05) and dp12 are capable of significantlyenhancing BMP-2-induced ALP activity over native heparin (p<0.01). Dp12salso enhanced osteogenic gene transcription, but not to the level ofnative heparin in the case of Alpl and Sp7 (FIG. 4 B, D). Thus theminimum oligosaccharide length required for maintenance of BMP-2-inducedtranscription of osteogenic genes and enhancement of ALP activity is adp12.

2.5. Selectively Desulfated Heparin Interactions with BMP-2

The structure of the disaccharide units in heparin is depicted in FIG.5A. Heparin chains contain an abundance of N-, 2-O-, and 6-O-sulfategroups, as well as the rarer 3-O-sulfate group, and these substitutions(when organized into specific sequences or domains) are responsible forinteraction with heparin/HS-binding proteins. To determine theimportance of specific sulfate groups of heparin/HS chains in thebinding of BMP-2, we performed SPR competitive binding experiments toascertain how well selectively desulfated heparins can out-competesurface heparin for the binding of BMP-2. Heparin and de-2-O-sulfatedheparin sequestered a substantial amount of BMP-2 into solution (73%) inSPR competition assays (FIG. 5B). As observed in the GAG binding plateassay, de-6-O-sulfated heparin displayed a marginally lower affinity toBMP-2, sequestering 63% of BMP-2 in solution (FIG. 5B). Finally,de-N-sulfated heparin was unable to bind and sequester any BMP-2 intosolution, however, re-N-acetylation of the free amine group rescuedBMP-2 binding, resulting in 51% of BMP-2 being sequestered into solution(FIG. 5B).

These data indicate a hierarchy of necessity, wherein N-sulfation isessential for BMP-2 binding, with 6-O-sulfation partially contributingand 2-O-sulfation only minimally.

2.6. Effect of Selectively Desulfated Heparins on the Thermal Stabilityof BMP-2

We further investigated the interactions of selectively desulfatedheparins and BMP-2 by using DSF. Pre-incubating BMP-2 with selectivelydesulfated heparins resulted in a decrease in the melting temperature ofBMP-2 compared to that with heparin, evident by a shift in peakfluorescence to a lower temperature (FIG. 5C). The loss of 2-O and6-O-sulfation resulted in a ˜10% and ˜20% reduction in the thermalstabilizing effect of heparin, respectively (FIG. 5D). Loss ofN-sulfation further reduced the thermal stabilizing effect of heparin,to ˜33% of that of native heparin (FIG. 5D). Re-N-acetylation ofde-N-sulfated heparin resulted in an improvement in the thermalstabilizing effect, from 33% to 71% (FIG. 5D). Statistical analysis viaone-way ANOVA revealed that sulfation contributes significantly to thethermal stabilizing effect of heparin toward BMP-2 (FIG. 5D;****p<0.0001). The loss of 6-O-sulfation results in significantreduction in stabilizing effect (***P<0.001), however the of2-O-sulfation did not result in a significant reduction in thestabilizing effect of heparin (ns-p>0.05). The loss of N-sulfationresulted in a significant reduction in stabilizing effect(****p<0.0001); subsequent re-N-acetylation improved the stabilizingeffect over de-N-sulfated species, but to a level that is stillsignificantly lower than that of native heparin (****p<0.0001). Thesedata once again confirm a hierarchy of importance with regards tosulfation. N-sulfation plays an essential role in the thermalstabilizing effect of heparin on BMP-2, while 6-O-sulfation offers apartial contribution and 2-O-sulfation offering a minimal contribution.

2.7. Effect of Selectively Desulfated Heparins on BMP-2-InducedOsteogenic Gene Transcription and ALP Activity

We next studied the importance of specific sulfate groups withinheparin/HS chains in BMP-2-induced Smad 1/5/9 phosphorylation,transcription of osteogenic genes and enhancement of ALP activity assay.Similar to observations with sized heparin oligosaccharides,phosphorylation levels of Smad 1/5/9 were not affected within 24 h byheparin and selectively desulfated heparins (FIG. 6Ai); however, at 48h. the levels decreased in cultures treated with de-N-sulfated orde-N-sulfated/re-N-acetylated heparins (FIG. 6Aii). After 72 h, culturestreated with heparin, de-2-O-sulfated or de-6-O-sulfated heparindisplayed the highest levels of Smad 1/5/9 phosphorylation (FIG. 6Aiii).Smad 1/5/9 phosphorylation was barely detectable in cultures treatedwith BMP-2 alone or BMP-2 with de-N-sulfated andde-N-sulfated/re-N-acetylated heparins after 72 h (FIG. 6Aiii). Heparinand selectively de-sulfated heparins alone had no effect on genetranscription (FIG. 6B-D) or ALP activity in C2C12 cells (FIG. 6E).However, when cells were treated with a combination of BMP-2 (100 ng/ml)and heparin (5 mg/ml), the induction of alpl, sp7 and runx2transcription and ALP activity was greatly enhanced (FIG. 6B-E).De-2-O-sulfation of heparin did not significantly affect the enhancementof alpl and runx2 transcription (FIG. 68 , C; p>0.05), but resulted in asignificant reduction in sp7 transcripts and ALP activity (FIG. 6D, E;p<0.0001). De-6-O-sulfation or de-N-sulfation and subsequentre-N-acetylation of heparin significantly reduced BMP-2-induced alpl andsp7 transcription and ALP activity (FIG. 68 -E); p<0.0001). These dataclearly indicate the essential role of N-sulfation in enhancingBMP-2-induced Smad 1/5/9 phosphorylation, osteogenic gene transcriptionand ALP activity in C2C12 cells, more so than 6-O- and especially2-O-sulfation.

2.8. Effect of Heparin Oligosaccharides on BMP-2-Induced OsteogenicDifferentiation and Mineralization

We further investigated the length and sulfation requirements withinheparin chains by employing an osteogenic differentiation assay over thecourse of 12 days. We observed that BMP-2 alone (100 ng/mL) was notsufficient to induce mineralization in C2C12 cells, as indicated bycalcium deposition detected by Alizarin red stain (FIG. 7A-B). Theaddition of heparin and BMP-2 lead to extensive mineralization, asevident from the presence of Alizarin red staining (FIG. 7A-B). Additionof heparin oligosaccharides dp4, dp6 and dp8 did not enhancemineralization in the presence or absence of BMP-2 (FIG. 7A-B). Additionof heparin dp10 with BMP-2 increased mineralization, although not to thelevel of heparin (p<0.0001), whilst addition of heparin dp12 enhancedmineralization to similar levels of native heparin (ns; p>0.05; FIG.7A-B). De-N-sulfated heparin was incapable of enhancing mineralization,and de-6-O-sulfated heparin only minimally enhanced mineralization (FIG.7A-B). De-2-O-sulfated heparin enhanced mineralization to the sameextent as native heparin (ns; p>0.05; FIG. 7A-B). Re-N-acetylation ofde-N-sulfated heparin partially rescued the ability of heparin toenhance BMP-2-mediated mineralization, but not to the level ofde-2-O-sulfated heparin (FIG. 7A). These data indicate that a minimumlength of a dp10 (optimally a dp12) and N-sulfation (optimally N- and6-O-sulfation) are required for heparin to enhance BMP-2-mediatedosteogenic differentiation in C2C12 cells.

2.9. Ectopic Bone Formation

The comprehensive in vitro data strongly suggests that lengths of atleast dp10 containing essential N-sulfated motifs are crucial for thebiological activation of BMP-2 by heparin. To widen the validity ofthese observations, selected oligosaccharides were next tested in an invivo rat ectopic bone-forming model. All rats survived the surgeries andrecovered uneventfully. PCL tubes were used in the study to allow forstraightforward implant retrieval and did not contribute to any boneformation (data not shown). FIG. 8 shows the combined results for thepositive and negative controls. The digital, 2D x-ray and 3D μ-CTmicrographs correlate well and demonstrated increased amounts ofmineralization when BMP-2 was present in the implant (Ctrl, BMP and Hop;FIG. 8A). Bone volume measurements revealed a similar trend (FIG. 88 ).BMP-2 alone resulted in ˜16-fold increase in bone volume compared tocontrol (Ctrl), while treatment with BMP-2 complexed with native heparinresulted in ˜0.2-fold increase over BMP-2 alone. One sample from theBMP-2/heparin group displayed no bone formation via x-ray and μ-CT,therefore was excluded from further analysis. The difference betweenBMP-2 and BMP-2/heparin groups is comparable to that observed in aprevious study using a similar ectopic model [49]. Paraffin and resinhistology were both performed to confirm the presence of bone-liketissue. No bone was detected in controls, unlike the BMP-2 andBMP-2/heparin treatments, which resulted in the infiltration of bonemarrow and the deposition of a calcified bone matrix (with osteocytesclearly visible within lacunae) as observed in H&E-, modifiedtetrachrome- and von Kossa/McNeals-stained sections (FIG. 8C).

Once the controls were established, the in vivo performance of BMP-2complexed with dp8, 10 and 12 saccharides were investigated (FIG. 9 ).Once again there was good correlation between the digital, 2D x-ray and3D μ-CT micrographs (FIG. 9A). One sample from each of the dp8 and dp10groups displayed no bone formation via x-ray and μ-CT, therefore theywere excluded from further analysis. The dp8 treatment resulted in lessbone deposition than dps 10 and 12. This trend was supported by bonevolume measurements that revealed a ˜1.7-fold increase with dp12 ascompared with dp8 (FIG. 9B). Histological sectioning confirmed thedeposition of a calcified bone matrix; this was interspersed within thebone marrow for all three groups, with the dp8 group displaying theleast overall amount of bone tissue (FIG. 9C). These data indicate aclear trend that increasing chain length enhances BMP-2-induced boneformation in vivo.

Finally, we contrasted the in vivo effectiveness of BMP-2 complexed withde-2-O-, de-6-O- and de-N-sulfated heparins (FIG. 10 ). One sample fromeach of the de-2-O-S and de-6-O-S groups displayed no bone formation viax-ray and μ-CT, therefore they were excluded from further analysis. Thedigital, 2D x-ray and 3D μ—CT micrographs showed that de-2-O- andde-6-O-sulfated oligosaccharides resulted in enhanced mineral depositioncompared to those de-N-sulfated (FIG. 10A). This observationcorresponded to bone volume measurements, where the former displayed anincrease in bone volume compared to the latter (FIG. 10B). Meanwhile,the loss of 2-O- and 6-O-sulfate groups did not affect the in vivoefficacy of these heparin variants, as indicated by comparable bonevolume measurements between the two groups. Histological sectionsmirrored this trend, and although calcified bone matrices with bonemarrow elements were observed for all treatments, the de-N-sulfatedgroup had noticeably reduced amounts of new bone tissue (FIG. 10C).

Example 3: Minimum Structural Requirements for BMP-2-Binding of HeparinOligosaccharides—Discussion

Information regarding the precise structure and composition of thebioactive domains of HS remains essential if reliable HS-basedtherapeutics are to be formulated. This knowledge, detailing thestructural and functional relationships between HS and proteins, mayalso aid in the eventual creation of synthetic HS analogues, which couldeventually negate the requirement for animal-derived products. The workhere describes our initial attempts to more closely define the essentialelements of BMP-2-activating heparin/HS domains, as we have previouslywith TGF-β1 [36]. The results of this study indicate a minimal chainlength of ten monosaccharides (dp10, decasaccharide), and the centralimportance of N-sulfation in the binding of BMP-2 and potentiation ofits biological activity, both in vitro and in vivo. These data regardingsulfation are in agreement with previous studies; these indicated ahierarchy of importance in heparin sulfation for effective binding ofBMP-2 and potentiation of its biological activity [37, 38].Specifically, data presented in these studies and the current studyhighlight the importance of N-sulfation. Such information on specificrequirements for size and sulfation may in turn be incorporated intoartificial HS analogues, as synthetic techniques begin to mature [39,40].

Here we first performed a competition assay to more systematicallydetermine the interaction between BMP-2 and various GAG subclasses,BMP-2 has been previously reported to strongly interact with heparin andHS GAGs. In our previous study, we observed little or no binding ofBMP-2 to CS-A, CS-C. CS-B (dermatan sulfate) or keratan sulfate (KS)[34]. Indeed, we further confirmed that CS-A, DS and KS bound littleBMP-2 and did not enhance BMP-2-mediated ALP activity in C2C12 cells(FIG. 11 ). Moreover, dextran sulfate, a large and highly chargedbranching polymer was able to bind BMP-2 with high affinity, butinhibited BMP-2 bioactivity in vitro (FIG. 11 ). Previous studies haveindicated that BMP-7 and FGF-1 interact with heparin and HS, but not CS,whereas FGF-2 can bind heparin and CS-E polysaccharides with similarbinding affinities [17, 41].

A number of heparin oligosaccharides of defined size were employed todetermine the importance of chain length on BMP-2 binding by SPRcompetition and size-exclusion chromatography assays. The resultsdemonstrated that heparin fragments of 3-4 disaccharides displayed alimited ability to bind BMP-2, and that binding gradually increasedthrough dp6 and dp8. Longer oligosaccharides, such as dp10 and dp12displayed high binding to BMP-2, but dp18 provided maximal binding,equivalent to that observed with heparin. Size-exclusion chromatographyresults confirmed the data acquired from SPR competition assays: dp12bound large amounts of BMP-2. We additionally performed DSF assays toestablish the importance of oligosaccharide length on the thermalstability of BMP-2. These data revealed that increasing chain lengthresulted in increased thermal stability of BMP-2, with a dp12 offering asubstantial increase in thermal stability over BMP-2 alone and a dp20offering thermal stability equivalent to that of heparin. Together,these data demonstrate a minimal requirement of a dp10, optimally adp12, and maximally a dp16 for the binding of heparin to BMP-2.

Key experiments have firmly established that a particular3-O-pentasaccharide within heparin is the minimal size required forantithrombin III binding and activity [28, 42-44]. In contrast, a dp8was the shortest heparin fragment that could subtend FGF-1 and FGF-2binding [45, 46], although dp10s were required for optimum proliferativeactivity [47, 48]. Similarly, the minimum size requirement for FGF-7 andFGF-10 binding to dermatan sulfate is a dp10, although largeroligosaccharides (dps14-20) are required for optimum activity [49. 50].In addition, we have previously confirmed that oligosaccharides of dp18and above are required for optimal binding of TGF-β1; furthermoreoligosaccharides of dp14 and above promote TGF-β1-induced biologicalactivity in human mesenchymal stem cells (MSCs) [36].

Previous studies have demonstrated that heparin is capable of enhancingBMP-2-induced osteogenic differentiation in C2C12 myoblasts in vitro[21]. Our data demonstrates that oligosaccharides of dp10 and above wereable to support this conversion, as evident by increases in osteogenicgene transcription, enhancement in ALP activity and induction ofmineralization. Most notably, the animal studies confirmed the in vitroresults, demonstrating that increasing chain oligosaccharide lengthmarkedly enhanced BMP-2-mediated bone formation in vivo.

We next determined the importance of specific sulfate groups withinheparin/HS chains BMP-2 binding by using chemically desulfated heparinpolysaccharides. A previous study has shown that rat MSCs cultured withde-2-O-sulfated heparin and BMP-2 displayed enhanced MSC proliferationand ALP activity compared to native heparin [38]. Here, N-sulfationproved critical for BMP-2 binding, whilst 6-O-sulfation was less so and2-O-sulfation had minimal impact on binding. Re-N-acetylation ofde-N-sulfated heparin partially rescued BMP-2 binding, but not to theextent of the other desulfated heparin species. Additionally,de-N-sulfated/re-N-acetylated heparin dp12s were incapable of bindingBMP-2 and offered little protection from thermal stress, and was unableto enhance BMP-2 bioactivity in vitro, further emphasizing the criticalrole of N-sulfation in heparin; BMP-2 interactions (FIG. 12 ). Thus, thecontribution of each sulfate group is not equal in the binding ofheparin/HS chains to BMP-2, implying that a particular sequence motifunderlies this phenomenon. A previous study indicated that a number ofvariants of desulfated heparin possessed similar net charge but differedsignificantly in their ability to protect BMP-2 from thermal stress[37]. These data again suggest that sequence and positioning of sulfategroups are essential in the binding of heparin to BMP-2. This result wassupported by enhancement of osteogenic gene transcription, ALP activityand mineralization in vitro and the in vivo studies where N-sulfationwas crucial for enhancing new bone formation. These results correlatewith previous studies showing that N- and 6-O-sulfates are moreimportant than 2-O-sulfates for binding to BMP-7 [17] and BMP-2 [37,38]. Interestingly, 2-N, 6-O-sulfated chitosan also displayed andability to enhance BMP-2 biological activity in vitro and in vivo,further indicating the importance of charge placement and density onpolysaccharide: BMP-2 interactions [51]. In contrast, the interaction ofHS with FGF-1 and FGF-2 require 2-O sulfate groups [31, 46]; althoughthe 6-O sulfate group is less important in binding FGF-2, thissubstitution plays a critical role in forming the FGF-2-FGF receptorcomplex, a step essential for FGF-2-mediated signaling [47, 48].

Interestingly, out of all treatment groups we observed the least in vivobone formation when BMP-2 was complexed with native heparin. This resultwas unexpected as complexes of BMP-2 and heparin leads to the greatestenhancement of in vitro osteogenesis. Additionally, a previous study byZhao and colleagues (2006) observed a significant increase in boneformation using identical doses of BMP-2 and heparin, albeit using adifferent ectopic model [22]. Despite these differences, theobservations are comparable to those made in a previous study by our owngroup using a similar ectopic model with identical doses of BMP-2 andheparin [32]. As eluded to in this previous study, the long-term effectsof heparin could differ substantially from the effects seen over shorterperiods. Furthermore, a study by Jiao and colleagues (2007) indicatedthat heparin can have an increasingly inhibitory effect on BMP-2bioactivity [52]. As previously mentioned, in vivo experiments performedin the study by Ratanavaraporn and Tabata (2012) suggestedde-2-O-sulfated heparin enhanced BMP-2-mediated bone formation overnative heparin [38]. Moreover, the heparin oligosaccharides andselectively desulfated heparins will possess a more uniform size (in thecase of the oligosaccharides) and sulfation pattern (in the case of thedesulfated heparins) than native heparin. This increase in homogeneitycould result in less off-target or inhibitory effects within the ectopicsite. Finally, the heparin oligosaccharides are a lower molecular weight(˜2400, 3000 and 3550 Da for dp8, dp10 and dp12 respectively) thannative heparin (˜15000 Da), decreasing the molar ratio between heparindp:BMP-2 in the ectopic site and potentially resulting in an increasednumber of oligosaccharide: BMP-2 interactions, thus enhancingBMP-2-mediated bone formation. This may also explain why we observe asignificant increase in ALP activity in C2C12 cells treated withBMP-2+dp12 versus BMP-2+heparin.

With the knowledge that heparin decasaccharides and de-2-O-sulfatedheparin can enhance BMP-2-mediated in vivo bone formation, the rationaldesign of osteoinductive biomaterials may be tuned and enhanced. Indeed,previous studies have noted that de-2-O-sulfation of heparin greatlydiminishes the molecules anticoagulant properties [53], which couldsubsequently lead to fewer off-target effects. Zieris and co-workers(2014) developed a novel starPEG/peptide/heparin hydrogel, usingdesulfated heparin to sequester growth factors of interest into thebiomaterial to enhance cell differentiation [54]. With knowledge ofminimum oligosaccharide lengths required for binding of various growthfactors and mitogens, such biomaterials could be further tuned toenhance specific interactions between proteins and scaffold, reducingunwanted side effects [55].

Although the details of the mechanism of action of the heparin fragmentsremain to be elaborated, it has been established that heparin acts toprolong BMP-2-induced cell signaling [22] as evidenced by thephosphorylation of Smad 1/5/9. Here we demonstrated that decasaccharidesand N-sulfate groups were necessary to potentiate these effects of BMP-2in C2C12 cells. Thus, we can conclude that BMP-2 preferences the bindingto N-sulfated heparin/HS domains of dp10 or greater over other GAGsubtypes. Such confirmatory information is an important step in therational design and chemical synthesis of HS-based drugs aimed atimproving bone tissue repair.

Example 4: Depolymerisation, Sizing and Compositional Analysis of HS-3and Fragments

4.1. Rationale

Data gathered in the heparin oligosaccharide/BMP-2 study (Examples 1-3)defined a minimal structural requirement for the effective stabilizationand potentiation of BMP-2's osteoinductive properties in vitro and invivo:

-   -   At least a decamer (dp10: five disaccharides)    -   NS>6OS>2OS—N-sulfation proved critical for BMP-2 binding, whilst        6-O-sulfation was less so and 2-O-sulfation had minimal impact        on binding    -   We therefore hypothesised that HS3 contained a BMP-2 binding        region of a certain size and sulphation    -   Due to the fundamental differences in the structure of heparin        and HS, we also hypothesised that this will be structurally        distinct from a heparin oligosaccharide of equivalent length.    -   Therefore, we aimed to depolymerise HS3 into pools of        oligosaccharides of various size and sulphation in a hope of        determining a number of potential structures which may        constitute the BMP-2 binding site within HS3.    -   Heparinase III was used for depolymerisation. Heparinase III        cleaves glycosidic linkages between GlcNAc/GlcNS and GlcA        residues therefore making it ideal for excising out the highly        sulphated domains of HS3.

4.2. Depolymerisation and Sizing

Summary

HS3 was enzymatically cleaved in transition regions(N-acetylated/N-sulfated domains) to liberate the highly charged andactive N-sulfated domains. 50 mIU of heparinase III was incubated with11 mg of HS3 for 24 h, followed by a second 50 mIU addition and 24 hincubation. Digested material was then passed through a 5 kDa molecularweight cut off (MWCO) filter, and the filtrate passed through a 3 kDaMWCO filer. The filtrate and retentate of each filter was then passedover a sephadex G25 column, and material eluting in the column voidregion (large material) was pooled. Material larger than 5 kDa was thenfurther fractionated by passing through a 10 kDa MWCO filter. Fractionswere weighed and re-suspended at 10 mg/mL and stored at −20° C. prior touse.

Depolymerisation Procedure

9 mg HS3 (batch 010), 6 mg HSpm (lot HO-10697) was reconstituted at 10mg/ml in LC/MS water, and total volume brought up to 900 μL. Then 100 μL10× heparinise buffer was added. Final salt concentration was 50 mMsodium acetate, 1 mM calcium acetate, pH 7

25 mIU heparinise III was then added to each sample and samples wereincubated at 37° C. for 24 h. A second addition of 25 mIU heparinise IIIwas then added to each sample and samples were incubated for further 24h at 37° C. Digestion was terminated by heating samples to 100° C. for 5minutes before cooling on ice. Samples were frozen and lyophilised todryness

Initial Sizing Step Procedure

Digested HS3 and HSpm were added to pre-washed 5000 Da MWCO Amicon ultracentrifugal filters (4 mL volume) and filtered by centrifuging for 30minutes at 4000 g. Retentate was kept, filtrate was added to pre-washed3000 Da MWCO Amicon ultra centrifugal filters (15 mL volume). Sampleswere filtered by centrifuging for 30 minutes at 4000 g.

Filtrate and retentate were pooled individually into groups: <3 kDa, >3k-5 Da, and >5 kDa

Desalting and Crude Second Sizing

Pooled oligosaccharides were desalted and crudely sized using a HiPrep26/10 desalting column. Superdrex peptide 10/300 gl analytical columnswere not used; trial runs resulted in minimal recovery of material,therefore different method was pursued.

Samples were loaded in 1 mL total volume followed by 1 mL water. In mostcases, a clear peak was observed at the void (FIG. 13 ), indicating thepresence of larger oligosaccharides; there were pooled and furtherreferred to as “large”. A broad peak followed, this was pooled as small.In the case of sub-3 kDa material, no sharp peak was observed; instead amuch broader peak appeared and crossed into the eluting salt peak. Thisis consistent with disaccharides. Peaks were pooled, lyophilised andweighed prior to reconstitution in water at 10 mg/mL.

Additional sizing—10 kDa Spin Filter

>5 kDa large HS3 and HSpm were added to pre-washed 10000 Da MWCO Amiconultra centrifugal filters (0.5 mL volume). Samples were filtered bycentrifuging for 30 minutes at 4000 g. Retentate and filtrate werefrozen at −80° C. and lyophilised to dryness.

Weights of Sized HS3

HS3>5-10 kDa—1.35 mg

HS3>10 kDa—0.64 mg

Samples were reconstituted at 10 mg/mL in deionised water and stored at−80° C.

4.3. Compositional Analysis of Sized HS3 Fragments

50 μg of each HS3 fragment was digested with 1 mIU of heparinase I, IIand III for 24 h at 37° C., followed by an additional 1 mIU of eachenzyme and a further 24 h incubation. Digests were terminated by heatingto 100° C. for 5 minutes, after which samples were frozen andlyophilised to dryness. Samples were re-suspended in 20 μL 0.1 M2-aminoacrydone in 85% DMSO/15% acetic acid and incubated for 20 min atroom temperature, protected from light. Samples were then reducedovernight with 20 μL 1M sodium cyanoborohydride. After reduction,samples were centrifuged at 13,000 rpm for 10 minutes and thesupernatant transferred to a fresh tube. The supernatant was diluted to400 μL with HPLC-grade water and passed through a 0.22 μm syringe-drivenfilter. Samples were analysed using a Dionex ICS-3000 high performanceliquid chromatography system and resolved using an Agilent ZorbaxEclipse XDB-C18 reverse phase chromatography column. Samples wereapplied to the column under 95% buffer A (5% buffer B (Acetonitrile) for1 min, before elution with a gradient of 5%-12% B over 29 min. Thecolumn was regenerated with 100% B for 5 min and re-equilibrated with 5%B for 5 min. Samples were detected with an RF2000 fluorescence emissiondetector (excitation wavelength—425 nm; emission wavelength—520 nm).Disaccharides were identified based on the elution points of knowndisaccharide standards and peak area corrected using pre-determinedcorrection factors based on variable labelling efficiency betweendifferent disaccharide species. Data was normalised as percentagecomposition and represents three HPLC analysis of an individual digestper sample.

Example 5: HS3 Fragments—Experimental Data

5.1. BMP2 Bioactivity Testing

C2C12 cells were seeded at 20,000 cells/cm2 overnight then treated thefollowing day with the sized heparin fragments, sized HS3 fragments,full length HS3 or full length heparin at 5 μg/mL with 100 ng/mL BMP2for 72 h. Total protein was extracted, quantified and ALP activitymeasured and represented as relative ALP activity versus BMP2 alone.

5.2. Enhancement of BMP2-Mediated ALP Activity

The data indicates that increasing chain length results in increased ALPactivity for both heparin and HS3 oligosaccharides. Unlike heparinfragments, which result in optimal enhancement of BMP2 activity at dp10and maximal at dp12, HS3 fragments are of at least dp18 and above inlength and optimally above dp36 (FIG. 14 ). This indicates that eitherthe active domains of HS3 are markedly larger, or HS3 requires more thanone active domain per chain to enhance BMP2 activity. These data revealimportant differences between the two molecules, and suggest that thedata generated in the manuscript will not significantly impact on workto elucidate the binding mechanism of HS3 to BMP2.

5.3. Compositional Analysis of HS3 Fragments

Compositional analysis reveals an obvious trend between length andoverall sulphation. The smallest fragments (below a dp12) contain thelowest degree of sulphation (FIG. 15 ), additionally they possess agreater amount of 2-O-sulphation than 6-O-sulphation, again correlatingwith data from the manuscript suggesting 6-O-sulphation is moreimportant than 2-O-sulphation in the binding of HS to BMP2. Fragments of18-36 saccharides display an ability to enhance BMP2-mediatedbioactivity, these also contain a higher abundance of sulphation and arecompositionally similar to the parental material (HS3 full length, FIGS.15 and 16 ). The greatest enhancement of BMP2-mediated bioactivity isinduced by fragments above 36 saccharides in length; these are also themost abundantly sulphated species, composed of ˜25% trisulphateddisaccharides and the highest proportion of N-sulphated, 6-O-sulphateddisaccharides (FIGS. 15-17 ). They also possess the lowest percentage ofunmodified disaccharides (table 1, 17.4%). These data suggest that N and6-O-sulphation are important for the enhancement of BMP2-mediatedbioactivity, which correlates with our hypothesis that 2-O-sulphation isthe least important modification for enhancing BMP2-mediatedbioactivity.

5.4. Compositional Comparisons Between Heparin Dp12 and HS3>Dp36

FIGS. 18, 19 and 20 compare the overall composition of a heparin dp12and HS3>dp36, which are the most biologically active fragments derivedfrom heparin and HS3, respectively. Structurally, each is distinctlydifferent; the heparin dp12 is predominantly composed of trisulphatedresidues (Table 2, 79%), whereas the HS3>dp36 contains more mono anddisulphated residues with 6-O-sulphation than heparin (14.48% 6-O and12.97% N-, 6-O vs 2.88% 6-O and 6.03% N-, 6-O, respectively). These dataon bioactivity and composition reveal important differences between thetwo molecules (HS3>dp36 and heparin dp12), and suggest that the datagenerated in the manuscript will not significantly impact on work toelucidate the binding mechanism of HS3 to BMP2.

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The invention claimed is:
 1. A biomaterial that is coated and/orimpregnated with an isolated heparin or heparan sulphateoligosaccharide, wherein the isolated heparin oligosaccharide has achain length of at least dp12 and no more than dp50, and wherein theisolated heparan sulphate oligosaccharide has a chain length of at leastdp36 and no more than dp50, and wherein the isolated heparin or heparansulphate oligosaccharide is capable of binding BMP2.
 2. The biomaterialof claim 1, wherein the biomaterial comprises a scaffold or matrixstructure.
 3. The biomaterial of claim 1, wherein the biomaterialcomprises hydrogel, fibrin, collagen, ceramic, metal, agarose, alginate,chitosan, polycaprolactone, poly(DL-lactide-co-caprolactone),poly(L-lactide-co-caprolactone-co-glycolide), polyglycolide,polylactide, polyhydroxyalkanoates, cellulose acetate; cellulosebutyrate, alginate, polysulphone, polyurethane, polyacrylonitrile,sulphonated polysulphone, polyamide, polyacrylonitrile,polymethylmethacrylate, hydroxyapatite, hyaluronic acid, an autograft,or an allograft biomaterial.
 4. The biomaterial of claim 1, wherein theisolated heparin oligosaccharide has a chain length of about dp12. 5.The biomaterial of claim 1, wherein the isolated heparin or heparansulphate oligosaccharide is N-sulphated, 6-O sulphated, and/or 2-Ode-sulphated.
 6. The biomaterial of claim 1, wherein the isolatedheparin or heparan sulphate oligosaccharide is a fragment of HS3.
 7. Thebiomaterial of claim 1, wherein the isolated heparin oligosaccharide hasa chain length of dp14 to dp50, dp16 to dp50, dp18 to dp50, dp20 todp50, dp22 to dp50, dp24 to dp50, dp26 to dp50, dp28 to dp50, dp30 todp50, dp32 to dp50, dp34 to dp50 and wherein the isolated heparin orheparan sulphate oligosaccharide has a chain length of dp36 to dp50,dp38 to dp50, dp40 to dp50, dp42 to dp50, dp44 to dp50, dp46 to dp50, ordp48 to dp50.
 8. The biomaterial of claim 1, wherein the isolatedheparin oligosaccharide has a chain length of dp12 to dp36, dp14 todp36, dp16 to dp36, dp18 to dp36, dp20 to dp36, dp22 to dp36, dp24 todp36, dp26 to dp36, dp28 to dp36, dp30 to dp36, dp32 to dp36, or dp34 todp36.
 9. The biomaterial of claim 1, wherein the isolated heparin orheparan sulphate oligosaccharide has a chain length of dp36 to dp50,optionally one of dp38 to dp50, dp40 to dp50, dp42 to dp50, dp44 todp50, dp46 to dp50, or dp48 to dp50.
 10. The biomaterial of claim 1,wherein the isolated heparin oligosaccharide has a chain length of dp18to dp40, optionally one of dp20 to dp40, dp22 to dp40, dp24 to dp40,dp26 to dp40, dp28 to dp40, dp30 to dp40, dp32 to dp40, dp34 to dp40,and wherein the isolated heparin or heparan sulphate oligosaccharide hasa chain length of dp36 to dp40, or dp38 to dp40.
 11. A method oftreatment, the method comprising the step of administering thebiomaterial of claim 1 to a subject in need thereof, wherein the methodof treatment comprises: a method of wound healing in vivo, the repairand/or regeneration of connective tissue, the repair and/or regenerationof bone, the repair and/or regeneration of bone in a mammal or a human,or the repair and/or regeneration of a broken bone.
 12. The method ofclaim 11, wherein the biomaterial that is coated and/or impregnated withan isolated heparin or heparan sulphate oligosaccharide is provided asan implant or a prosthesis.
 13. The method of claim 12, wherein theimplant is a bone graft substitute.
 14. The method of claim 11, whereinthe method of treatment is a method of treating of a bone fracture,degenerated bone, aging bone, diseased bone, and/or bone injury.
 15. Themethod of claim 11, wherein the method of treatment comprises the repairand/or regeneration of a long bone, short bone, flat bone, irregularbone, sesamoid bone, a skeletal bone, an appendicular bone, a bone ofthe pelvic skeleton, a bone of the cranio-facial region, a bone of theface, a bone of the mouth, a bone of the jaw and/or a bone of thevertebrae.
 16. The method of claim 11, wherein the isolated heparin orheparan sulphate oligosaccharide is administered during surgery.
 17. Themethod of claim 11, wherein the isolated heparin or heparan sulphateoligosaccharide is administered during dental, facial, or cranialsurgery.
 18. The method of claim 11, wherein the isolated heparin orheparan sulphate oligosaccharide enhances BMP2-mediated ALP activity.19. The method of claim 11, wherein the isolated heparin or heparansulphate oligosaccharide enhances BMP2-mediated Smad 1/5/9phosphorylation.
 20. The biomaterial of claim 1, wherein at least 80% ofN-acetyl-D-glucosamine (GlcNAc) residues in the isolated heparin orheparan sulphate oligosaccharide are N-sulphated.